The most common types of modification in human rRNA are pseudouridylation and 2′-O ribose methylation. These modifications are performed by small nucleolar ribonucleoproteins (snoRNPs) which contain a guide RNA (snoRNA) that base pairs at specific sites within the rRNA to direct the modification. rRNA modifications can vary, generating ribosome heterogeneity. One possible method that can be used to regulate rRNA modifications is by controlling snoRNP activity. RNA fragments derived from some small Cajal body-specific RNAs (scaRNA 2, 9 and 17) may influence snoRNP activity. Most scaRNAs accumulate in the Cajal body – a subnuclear domain – where they participate in the biogenesis of small nuclear RNPs, but scaRNA 2, 9 and 17 generate nucleolus-enriched fragments of unclear function, and we hypothesize that these fragments form regulatory RNPs that impact snoRNP activity and modulate rRNA modifications. Our previous work has shown that SMN, Drosha and various stresses, including etoposide treatment, may alter regulatory RNP formation. Here we demonstrate that etoposide treatment decreases the phosphorylation of SMN, reduces Drosha levels and increases the 2′-O-methylation of two sites within 28S rRNA. These findings further support a role for SMN and Drosha in regulating rRNA modification, possibly by affecting snoRNP or regulatory RNP activity.
The major types of modifications in human rRNA are pseudouridylation and 2′-O ribose methylation. Human rRNA contains around 100 of each of these modifications, which are performed by small nucleolar ribonucleoproteins (snoRNPs) (Darzacq et al., 2002; Khan and Maden, 1978; Maden et al., 1972; Maden and Salim, 1974; Lafontaine, 2015). SnoRNPs contain a guide RNA (snoRNA) that base pairs at specific sites within the rRNA to direct the modification. There are two kinds of snoRNPs: box H/ACA, which contain dyskerin and are responsible for the pseudouridylation of rRNA, and box C/D, which contain fibrillarin and perform ribosome methylation of rRNA (Kiss, 2004; Baserga et al., 1991; Fatica et al., 2000; Gautier et al., 1997; Schimmang et al., 1989; Szewczak et al., 2002; Tyc and Steitz, 1989; Watkins et al., 1996). Recent work, coupled with advances in the ability to detect pseudouridylation and 2′-O methylation modifications in a high throughput format, has shown that rRNA modifications can vary, generating ribosome heterogeneity (Birkedal et al., 2015; Lafontaine, 2015; Incarnato et al., 2017; Krogh et al., 2016; Sharma et al., 2017). The presence of a heterogenous pool of ribosomes may allow for the selective increase of a given 'type' of ribosome, leading to specialized ribosomes that are optimized for the translation of certain mRNAs (Lafontaine, 2015). Specialized ribosomes have recently been implicated as a major contributor to tumorigenesis (Marcel et al., 2015, 2013; Truitt and Ruggero, 2016).
One possible method that could be used to regulate rRNA modifications, and hence impact ribosome heterogeneity, is to control snoRNP activity. We have published that RNA fragments derived from some small Cajal body-specific RNAs (scaRNAs) may form regulatory RNPs (regRNPs) that influence snoRNP activity (Burke et al., 2018; Poole et al., 2017). As their name implies, scaRNAs accumulate in the Cajal body (CB), which is a subnuclear domain that takes part in the biogenesis of several different classes of RNPs, including small nuclear RNPs (snRNPs). Like rRNA, the small nuclear RNA (snRNA) component of spliceosomal snRNPs requires pseudouridylation and 2′-O ribose methylation modifications for full snRNP functionality (Darzacq et al., 2002; Tycowski et al., 1996; Kiss, 2004; Yu et al., 1998). These modifications in snRNA are guided by the scaRNA component of scaRNPs. (Darzacq et al., 2002; Kiss, 2004). Very interestingly, three scaRNAs (scaRNA 2, 9 and 17) generate nucleolus-enriched fragments of unclear function (Tycowski et al., 2004). We hypothesize that these RNA fragments, and other snoRNAs with uncertain roles, form regulatory RNPs that interact with the snoRNA component of snoRNPs and impact their activity. Therefore, by their interaction with snoRNPs, regRNPs modulate rRNA modifications (Burke et al., 2018; Poole et al., 2017).
Our previous work suggests that proteins enriched in the CB, such as coilin (the CB marker protein), SMN and WRAP53, impact scaRNA 2, 9 and 17 processing (Poole et al., 2016, 2017). SMN is the survival of motor neuron protein, which is mutated in most cases of spinal muscular atrophy and plays important roles in snRNP assembly (Coady and Lorson, 2011; Fischer et al., 1997; Meister et al., 2002; Paushkin et al., 2002; Pellizzoni et al., 1999, 2002). WRAP53 is a scaRNP/telomerase biogenesis factor that interacts with the Cajal body localization signal (CAB box) present in H/ACA scaRNAs and telomerase RNA (Richard et al., 2003; Jády et al., 2004; Mahmoudi et al., 2010; Tycowski et al., 2009; Venteicher et al., 2009; Zhu et al., 2004). In addition to these factors, we reported that Drosha may also contribute to the formation of regulatory RNPs (Logan et al., 2018). Drosha is a member of the RNase III family that initiates microRNA processing (Denli et al., 2004; Lee et al., 2003; Zeng et al., 2005). In the nucleus, Drosha enzymatically cleaves primary-miRNA (pri-miRNA) into the pre-miRNA stem/loop structure that is then transported to the cytoplasm for additional processing by Dicer (Bernstein et al., 2001; Grishok et al., 2001; Hutvagner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). Reduction of Drosha alters the fragment to full-length ratio of scaRNA 2 and 9, suggesting that scaRNA 2, 9 and 17 may be unorthodox substrates for Drosha (Logan et al., 2018).
Other conditions that may alter scaRNA 2, 9 and 17 processing are various stresses such as that induced by cisplatin or etoposide (Logan et al., 2018). Notably, we observed that the amount of the mgU2-30 fragment derived from ectopically expressed scaRNA9 is significantly reduced in cells treated with etoposide (Logan et al., 2018). In work presented here, we tested if a subset of rRNA modifications are altered by etoposide treatment. We also examined if scaRNA, snoRNA, SMN and Drosha levels were impacted by etoposide. These studies show that etoposide treatment significantly impacts the phosphorylation profile of SMN and reduces SMN interaction with coilin, resulting in gem formation. Etoposide was also shown to increase the 2′-O-methylation of 28S rRNA at sites 2388 and 3923, which was also found upon Drosha reduction. Collectively, our results demonstrate that stress conditions can influence rRNA modifications and suggest that these alterations may be mediated by changes in snoRNP or regulatory RNP levels.
Our previous results have shown that A2388 and G3923 in 28S rRNA and A484 in 18S rRNA may be subjected to regRNP control (Burke et al., 2018). In addition, we have also found that various stresses, including etoposide treatment, influence the relative amount of fragments derived from scaRNA 2 and 9 that we hypothesize form regRNPs (Logan et al., 2018). To examine if etoposide treatment impacts rRNA ribose methylation, we conducted primer extension assays using reverse transcriptase with low dNTP levels. Low levels of dNTP cause reverse transcriptase to pause near sites of ribose methylation (Maden et al., 1995). Fig. 1A is a representative primer extension assay with decreasing levels of dNTP showing the appearance of stop/pause signals as a consequence of 28S rRNA 2′-O-methylation at A2388 and C2352 when dNTP levels are 5 µM or 2.5 µM. RNA from untreated cells or cells treated for 48 h with 9 µM etoposide was then subjected to primer extension with low levels of dNTP to interrogate A2388 methylation. As shown in Fig. 1B, and quantified in Fig. 1C, the amount of 2388 methylation relative to the 2352 signal was significantly increased upon etoposide treatment compared to the untreated control. Primer extension with low dNTP was also used to evaluate 3923 ribose methylation in response to etoposide treatment (Fig. 1D,E). Like 2388, the amount of 3923 signal was increased (relative to 3904) with RNA from 48 h etoposide treated cells compared to RNA from untreated cells (Fig. 1D). This induction in 3923 signal was observed with 24 h, 48 h or 72 h etoposide treatment (Fig. 1E). In contrast, methylation of 18S A484 was not altered by etoposide treatment (Fig. 1F). Collectively, these findings suggest that 2′-O-methylation of rRNA is differentially affected by etoposide treatment.
Etoposide treatment increases the 2′-O-methylation of specific sites within 28S rRNA. (A) Primer extension using untreated RNA and a reducing amount of dNTPs, showing the induction of stop/pause signals corresponding to the 2′-O-methylation of 28S rRNA 2388 and 2352 at low (5 μM and 2.5 μM) dNTP concentrations. (B) Low dNTP primer extension using RNA from control or etoposide treated cells to evaluate 2388 methylation. HeLa cells were treated with 9 μM etoposide for 48 h in this experiment. (C) Quantification of 2388 methylation, normalized to the 2352 signal, relative to control, showing an increase in the relative amount of 2388 methylation in response to etoposide (n=3 biological repeats, *P<0.05). (D) Low dNTP primer extension using RNA from control or 48 h etoposide treated cells to evaluate 3923 methylation. (E) Quantification of 3923 methylation, normalized to the 3904 signal, relative to control, showing an increase in the relative amount of 3923 signal with 24, 48 and 72 h etoposide treatment (24 h and 72 h, n=3 biological repeats; 48 h, n=4 biological repeats. *P<0.05). (F) Low dNTP primer extension using RNA from control or 48 h etoposide treated cells to evaluate 18S rRNA 484 methylation. No significant difference was detected (n=3 biological repeats).
The increased 2′-O-methylation of 28S rRNA at A2388 and G3923 upon etoposide treatment may be the result of an increase in the snoRNP machinery that conducts these modifications. Additionally, it is also possible that regRNP levels, which are derived from selected scaRNAs, are impacted by etoposide. To indirectly determine the level of these RNPs, we evaluated that amount of the cognate RNA component by RT-qPCR. Using 5.8S rRNA as the normalizer, we first examined the level of scaRNA9 after treatment with etoposide for 24, 48 or 72 h. As shown in Fig. 2A, scaRNA9 is induced at all time points compared to that observed in untreated cells. When evaluating the levels of additional scaRNAs and selected snoRNAs after 48 h etoposide treatment, we found that all of these RNAs were induced relative to that obtained using control RNA from untreated cells (Fig. 2B). Notably, snord68 and snord111B, which are the guide RNAs for A2388 and G3923 2′-O-methylation, respectively, are increased by etoposide treatment. In contrast, U2 snRNA levels are not significantly impacted by this etoposide treatment. These findings suggest that the machinery required for the modification of snRNAs and rRNA is increased by this etoposide treatment. Furthermore, increased levels of scaRNA 2, 9 and 17 by etoposide may result in higher levels of regRNPs derived from these scaRNAs compared to that found in untreated cells.
Etoposide mediated increase of scaRNA and snoRNA. (A) Reverse transcriptase quantitative real-time PCR analysis of scaRNA9 in RNA from untreated or 24, 48 or 72 h etoposide (9 μM) treated RNA from HeLa cells. 5.8S rRNA was used as the normalizer and data are shown relative to control, which is set as 1 (n=3 biological repeats, *P<0.05). (B) Quantification of selected scaRNAs, snoRNAs and U2 snRNA from untreated RNA or RNA isolated from cells treated with etoposide for 48 h. 5.8S rRNA was used as the normalizer (n=3 biological repeats, *P<0.05). The data are shown relative to those obtained from untreated control RNA, which is set as 1. (C) Quantification of selected protein coding mRNA, including that from host genes which contain intron-encoded scaRNA9 (9-Host, CEP295) and scaRNA9-like (9-Like Host, EIF1AX). RNA from untreated control cells or cells exposed to 9 μM etoposide for 48 h was analyzed. 5.8S rRNA was used as the normalizer (n=3 biological repeats, *P<0.05). The data are shown relative to those obtained from untreated control RNA, which is set as 1. For A–C, error bars represent standard error about the mean. (D) Western blot of lysate obtained from untreated or 48 h etoposide treated cells. Antibodies to Drosha (top panel), coilin, SMN and beta-tubulin (bottom panel) were used.
In addition to non-coding RNA, we also examined the level of selected protein coding RNA in cells treated with 9 μM etoposide for 48 h (Fig. 2C). These messages include those mRNAs that produce Coilin, Dicer, Drosha, GAPDH and SMN. Relative to 5.8S rRNA, Coilin, Drosha, GAPDH and SMN mRNA levels are all reduced after etoposide treatment compared to that observed with RNA from untreated cells. Since Coilin, Drosha and SMN message levels were reduced by etoposide, we then examined protein levels from untreated and etoposide treated lysate. As shown in Fig. 2D, only Drosha showed a clear reduction in protein level by etoposide, consistent with the finding that Drosha mRNA was the most reduced of the RNAs we examined (Fig. 2C). Since scaRNA9 and scaRNA9-like are encoded in an intron of the host genes CEP295 and EIF1AX, respectively, we wanted to determine if the increased levels of scaRNA9 observed upon etoposide treatment is a consequence of an increase in the level of the RNA from the host genes. We found that CEP295 (9-Host) and EIF1AX (9-Like Host) mRNA were both reduced by etoposide treatment compared to that observed from control RNA (Fig. 2C). These findings suggest that the increase of scaRNA9 upon etoposide treatment (Fig. 2A,B) is not simply the result of induced host gene transcription.
It is known that interactions between coilin and SMN recruit the SMN complex to CBs (Boisvert et al., 2002; Hebert et al., 2001). Specifically, the post-translational modification of coilin by symmetrical dimethylation of arginines within the RG box of coilin mediates association with SMN, and the localization of the SMN complex to CBs. Although we did not observe a large decrease in the amount of SMN from etoposide treated cell lysate compared to control as determined by western blotting, we did detect a slight downward mobility shift (Fig. 3A, compare lanes 1 and 3 to lanes 2 and 4). In contrast, a mobility change was not detected for β-tubulin in etoposide treated cell lysate. (Fig. 3A, upper). We hypothesized that the mobility shift of SMN observed in etoposide treated cell lysate may be the result of decreased phosphorylation. To test this hypothesis, lysate from untreated cells was subjected to calf intestinal alkaline phosphatase (CIP) treatment. SMN in CIP treated lysate migrated more similarly to the SMN from etoposide treated lysate compared to the mobility of SMN from untreated lysate or no CIP control (Fig. 3B). These findings strongly suggest that SMN is hypophosphorylated by etoposide treatment. Because the mobility of SMN is only slightly affected on regular SDS-PAGE by phosphatase or etoposide treatment, we conducted SDS-PAGE using Phos-tag gels which are designed to exacerbate changes in mobility as a consequence of phosphorylation (Wako Chemicals USA, Richmond, VA,). As shown in Fig. 3C, CIP increases the amount of SMN present in a smaller mobility species (denoted by the A region) compared to untreated lysate, which contains larger mobility species that likely contain more phosphorylation (denoted by the B region). Quantification of the low or unphosphorylated SMN (A region) relative to phosphorylated SMN (B region) shows that etoposide treatment increased the relative amount of hypophosphorylated SMN (Fig. 3C,D). Moreover, treatment of cells with the phosphatase inhibitor okadaic acid (Lyon et al., 1997) slightly increased the relative amount of phosphorylated SMN (Fig. 3C,D). In particular, treatment of etoposide treated cells with okadaic acid resulted in a reduction in the amount of dephosphorylated SMN obtained with etoposide treatment alone (Fig. 3C,D). These findings reveal that SMN post-translational modification by phosphorylation is altered by etoposide treatment.
Hypophosphorylation of SMN by etoposide. (A) Western blot analysis of lysate from untreated or etoposide treated (9 μM for 48 h) HeLa cells. The blot was probed with antibodies to SMN (bottom) and beta tubulin (top). A slight downward mobility shift is seen in lanes 2 and 4. The estimated molecular weight of SMN (40 kDa) and beta tubulin (55 kDa) is shown. (B) Western blot to detect SMN using lysate treated with alkaline calf intestinal phosphatase (CIP) (lane 2). (C) Migration and detection of SMN using Phos-tag gels, which provide greater resolution of phosphorylated proteins compared to conventional SDS-PAGE. Low or hypophosphorylated SMN is indicated in the A region. More phosphorylated SMN is indicated in the B region. CIP treatment (lane 1) increases the amount of SMN in the A region, consistent with dephosphorylation. (D) Quantification of the signal in the A region divided by the signal in the B region for each condition tested, with the A/B ratio from untreated lysate set to 1. Etoposide treatment increases the amount of SMN in the A region relative to that in the B region by more than twofold compared to lysate from untreated cells (n=4 biological repeats, *P<0.05 compared to untreated, **P<0.05 compared to etoposide).
We have previously reported that etoposide treatment induces the dissociation of SMN from the CB, resulting in gem formation (Logan et al., 2018). For example, HeLa cells incubated with 9 μM etoposide for 48 h have numerous SMN foci lacking coilin (gems) and coilin foci lacking SMN (Fig. 4, right panel, arrowheads and double arrowheads). In contrast, untreated cells have CBs that are enriched for SMN and coilin (Fig. 4, left panel, arrows). To examine if the interaction between SMN and coilin was disrupted by etoposide, leading to gem formation, we conducted co-immunoprecipitation (Co-IP) assays using lysate from untreated or etoposide treated cells. Lysate was incubated with control mouse antibody or a mouse antibody to SMN, followed by capture of complexes on protein G beads, extensive washing, and SDS-PAGE/western blotting. Probing for coilin demonstrates that the amount of coilin Co-IPed by SMN is dramatically decreased in lysate from etoposide treated cells, compared to the amount of coilin recovered when using untreated lysate (Fig. 4B, upper panel, compare coilin signal in lane 6 to that in lane 5). Probing of the same blot for SMN shows that a large amount of SMN was immunoprecipitated from etoposide treated lysate (Fig. 4B, lower panel), and yet the amount of coilin recovered was reduced. These findings support the hypothesis that SMN hypophosphorylation as a consequence of etoposide treatment disrupts the interaction between SMN and coilin, resulting in gem formation.
Etoposide treatment induces gem formation and disrupts SMN interaction with coilin. HeLa cells were either untreated or treated for 48 h with 9 μM etoposide. The cells were then processed and SMN (red) coilin (green) and nuclei (DAPI, blue) were detected. Arrows indicate co-localization of SMN and coilin in CBs. Arrowheads denote gems, which are SMN foci lacking coilin. Double arrowheads mark coilin foci lacking SMN. (B) Co-IP of coilin by SMN is decreased by etoposide. Untreated or etoposide-treated lysate was subjected to IP with control (IgG, lanes 3 and 4) or SMN (lanes 5 and 6) antibodies. Complexes were recovered by protein G beads, which were then extensively washed, boiled, then run on SDS-PAGE followed by western transfer and detection of coilin (top) or SMN (bottom) using the appropriate antibodies. The input signal represents 2% of that used in the IP reactions.
Changes in nuclear organization by etoposide may underlie the observed alterations of 28S rRNA A2388 and G3923 2′-O-methylation. In particular, etoposide induced gem formation due to hypophosphorylated SMN could disrupt the normal trafficking of snoRNAs and scaRNAs, and possibly impact the formation of regRNPs. To test if we could antagonize the impact of etoposide on A2388 methylation, cells were incubated with the phosphatase inhibitor okadaic acid, alone or in combination with etoposide. RNA isolated from cells with these treatments was subjected to low dNTP primer extension to monitor A2388 methylation. As shown in Fig. 5, RNA from cells treated with okadaic acid and etoposide have significantly less A2388 methylation compared to RNA from cells treated with only etoposide (compare intensity of A2388 band in lane 2 to that in lane 4). Since we have previously shown that SMN phosphorylation is altered by etoposide and okadaic acid treatment (Fig. 3), it is possible that SMN phosphorylation is an important factor in the regulation of specific sites of rRNA 2′-O-methylation.
Etoposide-mediated induction of 28S rRNA 2388 2′-O-methylation is reduced by okadaic acid. (A) Low dNTP primer extension assay to analyze 2388 methylation using RNA from untreated cells or cells treated with 9 µM etoposide, 2 nM okadaic acid, and etoposide (9 µM)+okadaic acid (2 nM) for 48 h. (B) Quantification showing that etoposide+okadaic acid treatment decreases the relative amount of 2388 methylation compared to etoposide alone (n=4, P<0.05, * compared to untreated, ** compared to etoposide).
Unlike other scaRNAs, scaRNA 2, 9 and 17 can be processed to generate smaller nucleolus-enriched fragments of unclear function (Tycowski et al., 2004). One of these fragments is mgU2-30, which is derived from scaRNA9. We have proposed that these fragments form regulatory RNPs (regRNPs) that modify the activity of snoRNPs (Burke et al., 2018; Poole et al., 2017). We have also reported that various stress conditions, such as etoposide treatment, alter the ratio of the full-length scaRNA with the derived fragment (Logan et al., 2018). For example, using ectopically expressed scaRNA9, we found that etoposide treatment significantly decreased the amount of the mgU2-30 fragment relative to full-length scaRNA9 (Logan et al., 2018). In addition, etoposide treatment promotes SMN dephosphorylation, decreases coilin interaction and induces gem formation (Figs 3 and 4). Since the phosphatase inhibitor okadaic acid attenuates the etoposide mediated dephosphorylation of SMN (Fig. 3), we next examined if okadaic acid would alter the relative amount of the mgU2-30 fragment derived from scaRNA9. For this experiment, cells were transfected with a plasmid expressing scaRNA9, and treated 7 h later with 10 nM okadaic acid followed by an additional 17 h of incubation. RNA was isolated 24 h after transfection (17 h after okadaic acid treatment) and subjected to northern blotting and detection with a probe that anneals to the mgU2-30 fragment and full-length scaRNA9 (Fig. 6). We observed that, compared to RNA from untreated cells, the relative amount of the mgU2-30 fragment was increased by okadaic acid treatment approximately 1.7-fold. Hence the phosphatase inhibitor okadaic acid and etoposide, which decreases SMN phosphorylation, differentially impact scaRNA9 dynamics in regards to the amount of full-length versus processed fragment.
Okadaic acid alters the dynamics of full-length scaRNA9 and the mgU2-30 fragment. HeLa cells were transfected with scaRNA9 pcDNA 3.1+ for 24 h. 10 nM okadaic acid was added 7 h after transfection. RNA isolated from untreated and okadaic acid treated cells was then subjected to SDS-PAGE and northern blotting. ScaRNA9 and the mgU2-30 fragment were detected using a DIG labeled probe. Quantification was conducted using these and additional data by dividing the mgU2-30 fragment signal by the full-length scaRNA9 signal for each condition. The mgU2-30/full length scaRNA ratio for untreated cells was then set as 1. Okadaic acid increases the relative amount of the mgU2-30 fragment by approximately 1.7-fold (n=4 biological repeats, *P<0.05).
Our previous results have identified SMN and Drosha as factors that contribute to scaRNA 2, 9 and 17 dynamics (Logan et al., 2018). To examine if these proteins could be in the same complex, we conducted Co-IP experiments using FLAG-tagged DGCR8. DGCR8 is a well-described interactor of Drosha and is part of the Drosha complex. Cells transfected with a plasmid expressing FLAG-DGCR8 were lysed with KCl lysis buffer, followed by sonification and centrifugation. The lysate was then incubated with FLAG antibody (Flag) or control mouse antibody (IgG), followed by complex capture with protein G beads and washing of the beads with KCl lysis buffer. After SDS-PAGE/western transfer, the membrane was probed with antibodies to SMN (Fig. 7A, top panel), Drosha (middle panel) or FLAG (bottom panel). The amount of SMN recovered by the FLAG antibody is more than that recovered by the IgG control. Drosha and FLAG-DGCR8 are likewise found in higher amounts in the FLAG complexes compared to IgG control. These findings show that SMN can associate with the Drosha/DGCR8 complex. We next examined if endogenous SMN could Co-IP endogenous Drosha. For this experiment, cells were lysed in RIPA, which is a more stringent buffer than the KCl lysis buffer used above. RIPA lysate was incubated with control or SMN antibody, followed by complex capture on protein G beads, extensive washing and SDS-PAGE/western transfer. Probing of the blot with anti-Drosha antibodies showed that a faint signal was present in the reaction with anti-SMN but not in the control Ab reaction (Fig. 7B, upper panel, note faint signal for Drosha in lane 3). Re-probing of this same blot with anti-SMN verified that SMN was specifically recovered by the IP reaction containing anti-SMN (bottom panel, lane 3) but not recovered in the reaction with control Ab (lane 2). These findings show that endogenous Drosha and SMN can be found in a complex with one another, and possibly may contribute to scaRNP, regRNP and snoRNP biogenesis. Our previous finding that Drosha reduction alters the dynamics of scaRNA 2 and 9 processing (Logan et al., 2018) supports this hypothesis.
Drosha interacts with SMN and influences the modification of 28S rRNA A2388 and G3923. (A) SMN is associated with the Drosha complex. HeLa cells were transfected with FLAG-DGCR8, followed by lysis in KCl lysis buffer and IP with FLAG antibody (Flag) or control mouse antibody (IgG). After complex capture with protein G beads, beads were washed three times with KCl lysis buffer, followed by SDS-PAGE and western transfer. The membrane was probed with antibodies to SMN (top), Drosha (middle) and FLAG (to detect FLAG-DGCR8, bottom). Input represents 4.5% of the lysate used in the IP reactions. (B) Co-IP of endogenous Drosha with SMN. HeLa RIPA lysate was incubated with control antibody or SMN antibody, followed by complex capture with protein G beads. Beads were washed extensively then boiled and run on a SDS-PAGE followed by western transfer and detection of Drosha (top panel) or SMN (bottom panel) using the appropriate antibodies. A faint signal corresponding to endogenous Drosha is seen in lane 3, indicating that SMN and Drosha can form a complex. Reprobing of the same blot with SMN verifies the specificity of the reaction. Input represents 2% of that used in the IP reactions. (C) Low dNTP primer extension to detect 2388 methylation in RNA isolated from control siRNA or Drosha siRNA treated cells. An adjusted image is also shown to more easily visualize the increase in 2388 signal in the Drosha knockdown lane. Quantification was conducted by normalizing the 2388 signal to the 2352 signal and setting the control ratio value as 1. Drosha knockdown increases the relative amount of 2388 methylation by approximately 1.4-fold (n=3 biological repeats, *P<0.05). (D) Low dNTP primer extension to detect 3923 methylation in RNA isolated from control siRNA or Drosha siRNA treated cells. Quantification was conducted by normalizing the 3923 signal to the 3904 signal and setting the control ratio value as 1. Drosha knockdown increases the relative amount of 3923 methylation by a very small, but statistically significant amount (n=10 biological repeats, *P<0.05). (E) Drosha protein is reduced by Drosha siRNA. A western blot is shown. HeLa cells were transfected with negative control or Drosha siRNA for 48 h. The membrane was probed with Drosha antibody followed by probing with an antibody to β-tubulin.
To further implicate Drosha as a factor that impacts rRNA modification, the methylation of 28S rRNA A2388 and G3923 was examined by low dNTP primer extension using RNA from cells treated with control or Drosha siRNA. As shown in Fig. 7C, the methylation of A2388 was significantly increased in RNA from Drosha knockdown cells compared to control knockdown cells. G3923 methylation was also increased with Drosha knockdown (Fig. 7D), but not to the same extent as observed for A2388. Drosha knockdown by Drosha siRNA was verified by western blotting (Fig. 7E). Collectively, these results indicate that Drosha may be a component that helps to regulate rRNA 2′-O-methylation.
Ribosomes are not identical, but contain differences, such as variation in ribosomal protein complement and diversity of translation factors, that generate ribosome heterogeneity (Lafontaine, 2015). The major contributor to ribosome heterogeneity is rRNA modification, and the majority of these modifications are snoRNA-guided 2′-O methylations and pseudouridylations (Lafontaine, 2015). The ability to detect 2′-O methylation and pseudouridylation modifications in a high throughput format has given rise to the hypothesis of ribosome specialization (Birkedal et al., 2015; Sloan et al., 2017; Lafontaine, 2015; Incarnato et al., 2017; Krogh et al., 2016; Sharma et al., 2017). Additionally, increased rRNA 2′-O methylation as a consequence of upregulated fibrillarin has been implicated as a contributor to tumorigenesis (Marcel et al., 2015, 2013; Truitt and Ruggero, 2016). Since it is clear that all modification sites within rRNA are not equally modified in a pool of ribosomes (Lafontaine, 2015), a major goal of the rRNA field is to understand how rRNA modifications are regulated.
With this goal in mind, we have designed experiments to examine if a stress (etoposide treatment) known to impact the formation of regRNPs (Logan et al., 2018) disrupts rRNA modification. We also evaluated if etoposide treatment alters SMN and Drosha levels, which are two proteins we hypothesize are involved in the biogenesis of regRNPs (Logan et al., 2018). We have found that the 2′-O methylation of two sites within 28S rRNA, A2388 and G3923, are increased upon etoposide treatment but methylation of 18S rRNA A484 is not affected (Fig. 1). Etoposide treatment was also shown to induce selected scaRNA and snoRNA levels, but decrease selected mRNA levels, including that which encodes Drosha (Fig. 2). In addition to Drosha mRNA, Drosha protein levels were reduced by etoposide (Fig. 2D). Thus etoposide treatment increases the methylation of two sites (2388 and 3923) within 28S rRNA and is correlated with reduced Drosha levels. Interestingly, knockdown of Drosha by siRNA was also shown to increase 2388 and 3923 methylation (Fig. 7), supporting a role for Drosha in some capacity as a regulator of rRNA modifications. This hypothesis is further strengthened given that SMN and Drosha can form a complex (Fig. 7A). We are currently conducting in vitro studies to directly assess the role of Drosha in scaRNA 2, 9 and 17 processing.
Very interestingly, the phosphorylation of SMN was affected by etoposide treatment. Previous work has shown that SMN phosphorylation influences its localization and SMN complex activity, and the protein phosphatases PPM1G and PP1γ contribute to this process (Aoki et al., 2010; Burnett et al., 2009; Grimmler et al., 2005; Husedzinovic et al., 2015; Husedzinovic et al., 2014; Petri et al., 2007; Poole et al., 2017; Renvoisé et al., 2012). For example, nuclear SMN is hypophosphorylated compared to cytoplasmic SMN given that PPM1G is localized in the nucleus, and this hypophosphorylation is necessary for SMN accumulation in the CB (Petri et al., 2007). In our analysis of SMN protein levels obtained from cells treated with etoposide, we observed a slight downward mobility shift of SMN on standard SDS-PAGE followed by western transfer and detection consistent with dephosphorylation (Fig. 3A,B). Using Phos-tag gels, which have a greater resolution for phosphorylated proteins compared to standard SDS-PAGE, we observed that SMN is indeed more hypophosphorylated upon etoposide treatment compared to control (Fig. 3C). We also observed that the etoposide-induced hypophosphorylation of SMN is attenuated by the addition of the phosphatase inhibitor okadaic acid (Fig. 3C,D). Okadaic acid also blunts the increase of A2388 methylation observed in response to etoposide (Fig. 5) and alters the ratio of the mgU2-30 fragment to full-length scaRNA9 (Fig. 6). These findings suggest that SMN phosphorylation, which is influenced by etoposide and okadaic acid, may impact the regulation of rRNA modification. To more definitively prove the role of SMN phosphorylation in rRNA modification, additional studies utilizing phosphomimic and phosphonull SMN mutants will need to be conducted. Furthermore, the identification of the SMN phosphoresidues that are influenced by etoposide and okadaic acid treatment awaits further investigation.
In regards to nuclear organization, okadaic acid at higher concentrations than that used in our study has been shown to mis-localize CBs to the nucleolus (Lyon et al., 1997; Sleeman et al., 1998), demonstrating that nuclear organization is affected by hyperphosphorylation. We have shown that nuclear organization is also disrupted by etoposide (Gilder et al., 2011; Logan et al., 2018; Poole et al., 2017). Specifically, we have found that etoposide treatment (at 9 μM concentration) induces gem formation (Logan et al., 2018) (Fig. 4). Since etoposide results in SMN dephosphorylation and gem formation, we next tested if the interaction between SMN and coilin was disrupted in etoposide treated cells and observed that it was (Fig. 4B). These results show that SMN phosphorylation is a major contributor to gem formation and coilin interaction, as is the post-translational modification of coilin by symmetrical arginine dimethylation (Boisvert et al., 2002; Hebert et al., 2002, 2001).
Collectively, the data shown here support the hypothesis that various stress conditions which impact regulatory RNP biogenesis may alter rRNA modification. Our data also further strengthen the link implicating SMN and Drosha as contributors towards the generation and regulation of the rRNA modification machinery. Studies such as these will likely continue to reveal novel methods by which non-coding RNAs impact cellular metabolism. For example, a recent study on scaRNA2 demonstrated that this scaRNA promotes chemotherapy resistance by binding miR-342-3p (Zhang et al., 2018). It is probable that non-coding RNAs in the nucleolus packaged in regRNPs likewise interact with snoRNAs and thereby regulate snoRNP activity, resulting in ribosome heterogeneity.
HeLa cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in DMEM media (Invitrogen) supplemented with 10% heat inactivated fetal bovine serum (Gibco) and 1% penicillin streptomycin (Corning, Manassas, VA, USA). Cells were cultured in a 5% CO2 incubator at 37°C. Sca9 was ectopically expressed using the pcDNA3.1+ expression vector as previously described (Enwerem et al., 2014, 2015; Poole et al., 2017). FLAG-DGCR8 plasmid was obtained from Addgene (Watertown, MA, USA). For transfection of 60 mm dishes, 1 μg of plasmid was diluted in 97 μl Opti-MEM (Gibco) and 3 μl Fugene HD (Promega) was added and allowed to complex for 5 min before adding to cell culture. For experiments with drug treatments, cells were seeded a day in advance to be 70–100% confluent at time of treatment. Etoposide (Toposar, Teva Parenteral Medicines, Inc, Irvine, CA, USA) at 9 μM or Okadaic acid at 2 or 10 nM was added, depending on experiment, for 17, 24, 48 or 72 h. For siRNA transfections, RNAiMax was utilized (Invitrogen), and siRNAs are as described previously (Logan et al., 2018).
SMN forward (5′-GTG GTT TAC ACT GGA TAT GGA AAT AG-3′), reverse (5′-GAT TTA TTT CCA GGA GAC CTG GAG TTC-3′).
RNA was extracted from 24, 48 or 72 h treated or transfected HeLa cells with TRI-REAGENT (Cincinnati, OH, USA) according to the manufacturer's suggested protocol. 2 μg RNA was prepared with 1 μl Reverse Transcriptase buffer (New England Biolabs, Ipswich, MA, USA), 1 μl of 5 μM dig labeled primer designed to base pair downstream of the methylation site of interest (Integrated DNA Technologies, Coralville, Iowa, USA) and DEPC H2O to 8 μl. After 2 min at 95°C and 10 min at 42°C, 1 μl Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA) and 1 μl dNTPs were added and samples returned to 42°C for 1 h. The amount of dNTPs used are as noted, or were low concentrations (2.5 μM or 5 μM) used to detect ribose methylation. Samples plus loading buffer were run on a pre-warmed 15% TBE urea gel (Invitrogen) in 1× TBE at 180 V for 80 min. Gel was then rinsed in 1× TBE for 10 min. cDNA product was transferred to membrane using iBlot DNA transfer stacks (Invitrogen) with the iBlot Gel Transfer device (Life Technologies) using program 5 for 3 min, rinsed in ultrapure H2O and crosslinked at 120 K μJ/cm2. Membrane was incubated in Roche 1× blocking buffer for 15 min with slow rotation, then 30 min with slow rotation in Roche Anti-Digoxigenin-AP Fab fragments at 1:10,000 in Roche blocking buffer and washed with slow rotation in 1× wash buffer, (Roche wash and block buffer set, Roche, Mannheim, Germany). Membrane was developed with 1× CSPD in development buffer at 1:100 for 5 min at room temp, then placed between transparencies for 15 min in a 37°C incubator. Chemiluminescent images were captured and quantified with a Bio-Rad Chemi Doc Universal Hood and Quantity One Software (Bio-Rad, Hercules, CA, USA). Digoxigenin labeled DNA oligonucleotides were obtained from Integrated DNA Technologies (Coralville, Iowa, USA) and are as follows: 18S rRNA A484 site; 5′-DiGN/GCGCGCCTGCTGCCTTCCTTGGA-3′, 28S rRNA G3923 site; 5′-DiGN/CGCCGGGGGCCTCCCACTTATT-3′, 28S rRNA A2388 site; 5′-DiGN/CCCATGTTCAACTGCTGTTCAC-3′.
HeLa cells were lysed in RIPA buffer (50 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 0.25% Na-Deoxycholate, 1 mM EDTA, 0.1% SDS) plus Protease Inhibitor Cocktail (Thermo Fisher Scientific) and placed on ice. Cultures were collected into microtubes and sonicated briefly before centrifugation for 15 min at 4°C, 12,000 RPM. 10–15 μl of samples were run on SDS page using precast Bio-Rad 10% gels (Bio-Rad). For Co-IP experiments, lysate was incubated with 4 µg SMN or control mouse antibody for 1 h, followed by the addition of a protein G slurry and subsequent overnight incubation with mild shaking. The bead complexes were then washed five times with 1.5 ml per wash with RIPA plus Protease Inhibitor Cocktail, re-suspend in SDS-PAGE loading buffer, boiled, centrifuged and subjected to SDS-PAGE. Gels were run at 200 V for 55–60 min. Where noted, cells were lysed in KCL lysis buffer (20 mM Tris, pH 8.0, 100 mM KCl, 0.2 mM EDTA) followed by sonication and centrifugation as described above. Lysate was subjected to IP with 3 μg FLAG antibody or control mouse antibody. For these reactions, beads were washed three times with KCL lysis buffer before the addition of SDS-PAGE loading buffer. For Phos-tag gels, 7.5% precast zinc containing Phos-tag gels were obtained from Wako Chemical (Wako Chemicals USA, Richmond, VA). HeLa cells were lysed in RIPA buffer without EDTA. Phos-tag gels were electrophoresed in a cold room at 200 V for 55–60 min, followed by two 10 min rinses in 1× transfer buffer containing 10 mmol EDTA with gentle rotation and an additional 10 min rinse in transfer buffer alone. Transfer and detection of western blots were described previously (Poole et al., 2016). A Chemidoc system (Bio-Rad) was used to image the blots and adjustments to images were made using the transformation settings on QuantityOne software and applied across the entire image.
Antibodies used include: SMN, mouse/monoclonal (610646), BD Transduction Laboratories (San Jose, CA, USA); Beta tubulin, mouse/monoclonal (T5201), Sigma-Aldrich; Drosha, rabbit/monoclonal (D28B1), Cell Signaling; Coilin, rabbit/polyclonal (sc-32860), Santa Cruz Biotechnology; Control mouse IgG (sc-2025), Santa Cruz Biotechnology; FLAG, mouse/monoclonal (F3165), Sigma-Aldrich.
For dephosphorylation with CIP (New England Biolab, Ipswich, MA, USA), 5–10 μl of HeLa lysate was mixed with 1× NEB buffer 3 with 0.5–1.0 unit CIP/μg protein and DEPC H2O in 20 μl and incubated at 37°C for 60 min. Control reactions contained all of the above except CIP and were also incubated at 37°C for 60 min.
RNA was extracted from 48 h untreated or treated HeLa cells with TRI-REAGENT (Cincinnati, OH, USA) according to the manufacturer's suggested protocol. Equal volume of gel loading buffer was added to 10–16 μg of samples and then heated at 95°C for 5 min. RNA was run on a 6% denaturing polyacrylamide gel (Invitrogen) in 1× Tris-Borate-EDTA (TBE) at 200 V for approximately 30 min. After a 10 min wash in TBE, RNA was transferred to membrane with iBlot DNA transfer stacks (Invitrogen) and iBlot Gel Transfer device (Life Technologies, Grant Island, NY, USA) using program 5 for 5 min. Membrane was rinsed in ultrapure water then dried and crosslinked using a UV cross-linker (UVP, Upland, CA, USA) at a setting of 120,000 μJ/cm2. The membrane was then pre-hybridized in a hybridization bottle using 15 ml of Ultrahyb Ultrasensitive Hybridization buffer (Ambion Life Technologies, Grand Island, NY, USA) for 30 min at 42°C in a rotating hybridization oven. The membrane was then probed overnight with a DIG-labeled DNA oligo probe, which hybridizes to full-length scaRNA9 and the mgU2-30 fragment, as described elsewhere (Logan et al., 2018). Membranes were then prepared for detection using the DIG Wash and Block kit (Invitrogen) following the manufacturer's suggested protocol with the Anti-DIG antibody used at 1:10,000. Detection was carried out using CSPD (Roche, Mannheim, Germany) following the manufacturer's suggested protocol. Blots were imaged using a Chemidoc imager (Bio-Rad). Adjustments to images were made using the transformation settings on QuantityOne software and applied across the entire image.
IF, image capture and processing were conducted as previously described (Logan et al., 2018). Briefly, cells were seeded into chambered slides and untreated or treated with etoposide (9 μM) for 48 h. Cells were then fixed with paraformaldehyde, followed by extraction with triton and blocking with normal goat serum. Anti-SMN and anti-coilin antibodies (described above) were then used, along with the appropriate secondary antibodies. DAPI was used to stain the nucleus.
Student's t-test was performed to determine statistical significance, *P≤0.05.
Conceptualization: M.F.B., M.K.L., M.D.H.; Methodology: M.F.B., D.M.M., M.K.L., M.D.H.; Validation: D.M.M., M.D.H.; Formal analysis: M.F.B., D.M.M., M.K.L., M.D.H.; Investigation: M.F.B., D.M.M., M.K.L., M.D.H.; Resources: M.F.B., M.K.L., M.D.H.; Data curation: M.F.B., D.M.M., M.K.L., M.D.H.; Writing - original draft: M.F.B., M.D.H.; Writing - review & editing: M.F.B., D.M.M., M.K.L., M.D.H.; Visualization: D.M.M., M.K.L., M.D.H.; Supervision: M.D.H.; Project administration: M.D.H.; Funding acquisition: M.D.H.
This work was supported by internal funds from the Department of Cell and Molecular Biology, The University of Mississippi Medical Center, Jackson, MS, USA.
Albaret, M. A. et al.
(1996). Elements essential for processing intronic U14 snoRNA are located at the termini of the mature snoRNA sequence and include conserved nucleotide boxes C and D. RNA 2, 118-133.


The Z1 is a natural, simple and beautiful handmade bamboo pendant light, making unique and fresh to any interior.
With it's design based upon a chinese lantern this oversized pendant light has a cool cotton shade tied tautly to it's lightweight bamboo frame. The light is supplied complete with a black metal ceiling rose, black fabric flex and a white ceramic bulb holder. Finish the look with a large globe carbon filament bulb.


A second case of possible measles exposure has been confirmed at Los Angeles International Airport.
LOS ANGELES (KABC) -- Los Angeles International Airport travelers are being warned of possible measles exposure again after a second confirmed case traveled through the airport.
A person with the viral infection passed through the airport in Terminal 7 from 10 a.m. to 12:30 p.m. on March 5, arriving on United Airlines Flight 240.
That person traveled throughout Los Angeles County while infectious, including at Enterprise Rent-A-Car at 1719 Wilshire Boulevard in Santa Monica on March 6.
Health officials worry many could have been exposed.
Symptoms include cough, fever, runny nose, red eyes and a rash, which usually appears 10 to 21 days after exposure. If you have those symptoms, you're urged to contact your healthcare provider immediately.
Another person traveled through LAX with measles on Feb. 21. Health officials don't believe the two cases are related.
More information about measles from the county is available here.


text:
Hednotodes är ett släkte av fjärilar. Hednotodes ingår i familjen mott.

Kladogram enligt Catalogue of Life:

Källor

Mott
Hednotodes

redpajama_set_name: RedPajamaWikipedia


text:
<?php
// src/AppBundle/Controller/RecipeController.php

namespace AppBundle\Controller;

use Sensio\Bundle\FrameworkExtraBundle\Configuration\Route;
use Sensio\Bundle\FrameworkExtraBundle\Configuration\Method;
use Sensio\Bundle\FrameworkExtraBundle\Configuration\ParamConverter;
use Symfony\Component\HttpFoundation\Request;
use Symfony\Component\Debug\ExceptionHandler;

use AppBundle\Domain\Recipe;
use AppBundle\Domain\Ingredient;
use AppBundle\Services\RecipeService;
use AppBundle\Controller\BaseJsonResponse;
use AppBundle\Repository\IngredientNormalizer;
use AppBundle\Repository\RecipeNormalizer;

Class RecipeController extends BaseJsonResponse{

	const RECIPE_SERVICE = 'recipe.service';

	public function __construct(){
		parent::__construct();
	}

	/**
	 * @Route("/recipe/{id}", name="getRecipe")
	 * @Method({"GET"})
	 */
	public function getRecipe($id){
		$recipeService = $this->get(self::RECIPE_SERVICE);
		$recipe = $recipeService->getRecipe($id);
		return $this->writeJson($recipe);
	}

	/**
	 * @Route("/recipe/{id}", name="saveRecipe")
	 * @Method({"POST"})
	 */
	public function saveRecipe($id, Request $request){
		$normalizer = New RecipeNormalizer();
		self::getRecipe($id);

		$recipeService = $this->get(self::RECIPE_SERVICE);
		$data = json_decode($request->getContent(), true);

		$recipe = $normalizer->normalize($data);

		try{
			$newrecipe = $recipeService->updateRecipe($id, $recipe);
			return $this->writeJson($newrecipe);
		}catch(Exception $e){
			http_response_code(400);
		}
	}

	/**
	 * @Route("/recipes", name="getRecipeList")
	 * @Method({"GET"})
	 */
	public function getAllRecipes(){
		$recipeService = $this->get(self::RECIPE_SERVICE);
		$recipes = $recipeService->getAllRecipes();
		return $this->writeJson($recipes);
	}

	/**
	 * @Route("/recipe/create/{name}/{description}/{course}", name="createRecipeAction")
	 * @Method({"GET"})
	 */
	public function createRecipeAction($name, $description, $course){
		$recipeService = $this->get(self::RECIPE_SERVICE);
		$recipe = $recipeService->createRecipe($name, $description, $course);
		return $this->writeJson($recipe);
	}

}

redpajama_set_name: RedPajamaGithub


Over the last many years, I've operated from a framework of probability requiring me to never believe anything with certainty; I still do this, seeing it as part of radical empiricism; I see reality as the only kind of truth, and any other things we generally call truth to only be so by analogy with a weaker claim to the term. I consider everything I call true to have a slight hedge to it; no proofs, no absolute solidity. If it is meaningful to judge it as truth, it must be falsifiable, and there is always some way, however unlikely it seems, by which I might be forced to give it up; the chances of this are practically (but not theoretically) identical to how likely I think it to be true.
I've been working to construct a doubting part of myself with a different perspective to challenge me with on this point; essentially a stronger form of the position that would fudge nearly-certain things to certainty. Perhaps without the ability or belief in reasoning with infinitely-grained probability statistic. If someone were to say "I am certain in $belief", and if pressed on it they said "I would revise if presented with contrary evidence, but it seems really really unlikely to me", I'm not sure how I would criticise that perspective. It's not Bayesian, but then I don't accept much of the philosophical trappings that normally go with Bayesian thinking (I consider frequentist reasoning to be generally stronger-when-available, although this is a complex topic that I think I covered in a previous entry). My gut instinct is to say "that's not how probability works", but pitting my definition against the one I'm talking about here doesn't feel like a clear win either.
Maybe I should say that it's a viable perspective, but weaker in predictive power (in the rare areas where it differs, to the extent of the size of the fudging it does) than my framework? I'm almost nervous about arguing against it because it easily falls into the messy realm of the role of judgement in statistical reasoning, and while we can capture *some* of that kind of thing through nicer mechanical reasoning methods (SVDs and similar), so much of the areas where we want predictive power (even if weak) is hard to quantify; the conversation (inner though it is) might easily fall into that area.


text:
Гле́ндейл () — англизированная версия гэльского Gleann Dail, означающего «долина плодородной, низколежащей, пахотной земли».

 Глендейл — город в штате Калифорния, США.
 Глендейл — город в штате Аризона, США.
 Глендейл — город в штате Висконсин, США.
 Глендейл — город в штате Миссури, США.
 Глендейл — город в штате Колорадо, США.
 Глендейл — деревня в штате Огайо, США.
Глендейл — город в штате Орегон, США.


text:
Lobophyllia costata är en korallart som först beskrevs av James Dwight Dana 1846.  Lobophyllia costata ingår i släktet Lobophyllia och familjen Mussidae. Inga underarter finns listade i Catalogue of Life.

Källor

Stenkoraller
costata


text:
Tag: Rescuers
Disney Double Blu-ray Giveaway
August 31, 2012 September 9, 2013 Chip
We love connecting with our fans and other Disney Bloggers. We have been blogging now for 5 years and constantly meeting new people. Today we
Blu-ray Disc, Disney Contest, Disney Giveaway, Down Under, Dumbo, DVD, Rescuers, Sunday, The Walt Disney Company, Twitter
'The Rescuers: 35th Anniversary Edition Double Feature' Blu-ray Review
August 22, 2012 September 9, 2013 Chip and Co Past Writers and Friends
For me, 'The Rescuers' series is one of the true gems of Walt Disney Animation's filmography. Both movies are full of colorful characters, exciting adventure,
animation, Bernard, Bianca, Blu-ray Review, Bob Newhart, disney, Eva Gabor, George C. Scott, John Candy, Movie Review, Rescuers, Rescuers Down Under, The Walt Disney Company, Walt Disney Animation
Coming to Disney Bluray and DVD for 2012
July 29, 2012 September 9, 2013 Chip and Co Past Writers and Friends
Here's a look at the box art for some upcoming Walt Disney Home Entertainment animation releases – including Disney Secret of the Wings, The Tigger
Aristocats, Art, Avengers, Blu-Ray, Cinderella, DVD, Finding Nemo, Lady and the Tramp, Marvel, Pixar, Pocahontas, Prep & Landing, Rescuers, Tigger, Tinker Bell, Walt Disney Studios Home Entertainment, Winnie the pooh

redpajama_set_name: RedPajamaCommonCrawl


text:
This is a blog of movie reviews written by Jake Mascotte. All rights reserved.
None, Action, Animation, Comedy, Commentary, Documentary, Drama, Miscellaneous, Mystery, Romantic Comedy, and Thriller
[0-9] a b c d e f g h i j k l m n o p q r s t u v w x y z
or by Title
February, 2018, Drama
Interpreting writer/director Paul Thomas Anderson films presents a challenge to movie audiences; even the seemingly straight forward Hard Eight, his directorial debut, came marinated in a point of view which was to resurface again and again his later work. From Boogie Nights through Magnolia, Punch Drunk Love and The Master to the horrifyingly brilliant There Will Be Blood, Anderson worldview seems permeated by a special appreciation of (if not reverence for) the often irrational, mystifying behavior of certain characters that emerge from his scripts. That tendency is on full display in his latest effort, but with more benign results.
Daniel Day Lewis delivers another mesmerizing performance here as Reynolds Woodcock, a brilliant and successful designer of couture clothing in mid-50's London. In partnership with his glacially-composed sister Cyril (Leslie Manville), Woodcock creates elegant dresses for elegant women who return year after year to be dressed, flattered and browbeaten by Woodcock's imperious manner and maniacally obsessive commitment to his talent. He's surrounded by women and impervious to their allure - - until he's captivated by Alma, (Vicky Krieps) a waitress who serves him one morning at a small restaurant near his country home. He finds her quiet, bemused manner and uncomplicated freshness appealing, first as a model, then muse, then lover. How will this haughty, sophisticated control freak adjust to life with someone so appealing uncomplicated?
But Woodcock's soon frustrated by Alma's unwillingness to bow to his will. Cyril has always catered to his idiosyncrasies, so why should Alma be different? His annoyance sours into petulant anger and it takes a sudden illness to shake Woodcock's arrogant self-confidence. He mellows, proposes and they wed, but soon Woodcock lapses back into mean-spirited self-absorption. Only a subsequent illness hints at a way forward and the film ends with an unspoken but tentative hopefulness.
The performances of Thread's female leads are remarkable. Cyril's Oscar-nominated icy cordiality counterbalances Alma's insouciance at every turn and their differing responses to Woodcock's brand of domestic tyranny create an understated tension that sustains the storyline from start to finish.
Oddly enough, Lewis' performance may be off-putting, epitomizing as it does that brand of artistic brilliance unable to recognize the difference between the legitimate conditions required by a creative personality vs. the egoistic demands of someone whose gifts have grown inherently self-absorbed. Lewis insists that this is his last film and if that turns out to be true, moviegoers will be deprived of the finest actor of his generation. As he nears his 51st birthday, lets hope this 3-time Oscar winner doesn't deprive us of another decade or more of compelling work.
In every detail from cinematography to production values to a musical score that enlivens the movie's action, Phantom Thread deserves the half-dozen Oscar nominations it's received for directing, acting, musical score, costume design and best picture.
The Verdict? A small gem whose off-putting premise isn't likely to be widely seen or admired.
Jake's Takes comments powered by Disqus


In the early 2000s, advances in technology and distribution made possible a vibrant new wave of Jewish and Israeli films. Around the same time, films started making appearances at major academic conventions, as scholars recognized their importance as teaching tools. In 2007, in light of these developments, AJS held our first film festival, initiated by Bernard Cooperman. Later, several film scholars came together to form the Film Committee to select films, negotiate with distributors, and organize film events.
Over the years, the AJS film program has evolved considerably. While initially we screened only modest documentaries, we now show some of the most exciting films coming out of the Jewish world. Still, we found that not enough attendees had a chance to see the films during the day. There was also no forum to address pedagogical applications of film. As a result, this year the Film Committee is pleased to introduce the next stage in our evolution—a formatting change that we believe will significantly improve attendees' experience of the film program.
The film program will now consist of two separate parts. The first part will be evening screenings of films of significant interest and entertainment value to the AJS constituency. The second part will be a pedagogical session on teaching with film.
Each evening screening will be introduced by a relevant scholar. At the upcoming AJS in Boston, the program will include: 18 Voices Sing Kol Nidre, a US documentary that tells the prayer's rich story; The Guardians of Remembrance, a new documentary filmed in Belarus, commemorating the Holocaust victims in the former Soviet areas; and two Israeli feature films: A Borrowed Identity, a new Israeli drama delving into the issues surrounding Palestinian identity in Israel; and The Dove Flyer, a rare tribute to the Jewish community of Iraq. The Kol Nidre documentary is introduced by a musician and singer, Jewlia Eisenberg, and the other films by scholars in the field. Keeping up with the task of providing AJS participants with materials for their own programming, we include distributor information for every film, including contacts for obtaining public performance rights.
For the pedagogy session, the Film Committee collaborated with the Pedagogy Division to develop a panel on teaching with film, where committee members will showcase both new films and their teaching methods. We will focus our inaugural session on pedagogical opportunities and strategies for using film to teach about the Holocaust. The session will consist of hands-on, practical demonstrations suggesting ways in which to select and present a film clip and facilitate classroom discussion. Each presenter will follow the same format: a brief introduction to the subject and the film, followed by a film clip, and a 10-minute discussion modelling a classroom discussion. Presentations will conclude with recommendations for accompanying readings and assignments. The goal of the session is to provide participants with a useful toolbox for instruction in the field of Jewish history and culture, Film Studies, and Hebrew.
The session will progress chronologically, opening with a presentation on The Unvanquished (1945, USSR), one of the first Holocaust films worldwide. The presenter, Olga Gershenson, will share her experience in using this film to teach about the history of the Holocaust in the occupied Soviet Union. The screening of a film excerpt (available online with English subtitles), will be followed by a model classroom discussion. Questions for discussion will include historical context of the events, modes of representation, and challenges to the mediation of the Holocaust in the Soviet Union.
The next presentation will focus on one of the films in the series The Decalogue: VIII – Thou Shalt not Bear False Witness (1989, Poland) by Krzysztof Kieslowski. This film is among the earliest Polish cinematic representations of the Holocaust, and raises questions about Polish non-Jewish attitudes towards Jews and the moral ambiguities of behavior during the war. The presenter, Sara R. Horowitz, will discuss how the film can be used in complicating discussions of rescue behaviors during the war, and the evolution of Polish wartime memory, placing it in the context of later Polish films about rescue.
Fateless (Hungary, 2005) is a focus of a presentation by Catherine Portuges. She will examine an excerpt from this story of a Hungarian Jewish adolescent deported to Auschwitz, and his return from the camps. Among the topics for discussion are: the language and style of the trauma representation; alienation of survivors; Hungarian responses to film, as well as current debates on antisemitism in Hungary.
The session will conclude with a discussion of an Israeli documentary Six Million and One (2011), presenting a family journey to retrace their father's remarkable life during the Holocaust based on his diary which they found after his death. The presenter, Dalit Katz, will explore the possible uses of the film in Hebrew classes, including discussion of colloquial Hebrew and explanation of Israeli cultural concepts.
We hope that you will join us both for the film screenings and for our first ever pedagogy session, and provide us with feedback. What we are most interested in hearing is what movies you want to see and in which formats.
Olga Gershenson is chair of the AJS Conference Film Committee and professor of Judaic and Near Eastern Studies at the University of Massachusetts-Amherst.


text:
IMPORTANT: For news articles and the latest updates surrounding the COIVD-19 Pandemic, log in and check the COVID-19 Critical Resources page.
January 2023 Impact, "Yellow," Now Online
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Report on Bipartisan Therapy Bill "SMART Act" Paves the Way for Congressional Action
New Report Identifies Potential Medicare Cost Savings by Changing Supervision Requirement; Underscores Critical Role of Therapy Assistants in Rural and Underserved Areas
Alexandria, VA – The results of a new independent, nonpartisan study, commissioned by a coalition of health care provider groups, show Medicare could save between $168 million and $242 million over 10 years by reducing administrative burdens on physical therapists and occupational therapists. These savings could help offset urgently needed changes necessary to improve access to therapy services for Medicare beneficiaries and to provide relief to rural and medically underserved areas of the country experiencing therapy workforce shortages. The win-win findings underscore the urgent need for Congress to pass pending legislation that would make those improvements possible.
The study, conducted by Dobson DaVanzo & Associates, looked at the potential financial impact of the Stabilizing Medicare Access to Rehabilitation and Therapy Act, or SMART Act (H.R. 5536), introduced in the U.S. House of Representatives by Rep. Bobby Rush, D-Ill., and Rep. Jason Smith, R-Mo. The bill aims to exempt rural or underserved areas from a 15% Medicare payment cut implemented earlier this year when services are provided by an occupational therapy assistant or physical therapist assistant. That cut was implemented on Jan. 1, 2022, and applies to OTA and PTA services under Medicare Part B.
"Many seniors and Medicare recipients rely on physical and occupational therapy services to maintain their independence and stay healthy and mobile after illness or injury," said Rep. Bobby Rush, D-Ill. "These misguided pay cuts will cause severe harm to older Americans in underserved communities and to physical and occupational therapy assistants — many of whom are people of color."
"Through the SMART Act we can improve access to physical and occupational therapy services while cutting red tape in Washington," said Rep. Jason Smith, R-Mo. "I'm proud to co-lead this legislation with Representative Rush that will not only expand seniors' access to treatment, especially in rural and underserved areas, but will also preserve the functional therapy community for future generations of Medicare patients."
In addition, the legislation would bring supervision requirements of OTAs and PTAs under Medicare Part B in private therapist practices in line with state licensure laws, as well as all other settings where Medicare beneficiaries receive therapy services.
The study projects costs and savings associated with both provisions of the SMART Act. Results show that the cost savings generated through the change in supervision for OTAs and PTAs in the outpatient setting could help pay for the elimination of the 15% assistant payment cut in rural or underserved areas, which is estimated to cost CMS from $741 million to $1.58 billion over 10 years.
These provisions of the SMART Act would make therapy services more accessible to millions of Americans experiencing challenges accessing these services in rural or underserved areas, where beneficiaries are 50% more likely to receive therapy from an OTA or PTA. The standardization of general supervision requirements for private practices is cost-effective and would reduce administrative burden while implementing common-sense consistency with state laws and across all other Medicare settings.
The research was commissioned by the American Physical Therapy Association, American Health Care Association, American Occupational Therapy Association, Alliance for Physical Therapy Quality and Innovation, National Association of Rehabilitation Providers and Agencies, National Association for the Support of Long-Term Care, and APTA Private Practice, a section of the American Physical Therapy Association. The report can be accessed online at: Dobson | DaVanzo & Associates, LLC - Health Care Consulting - Publications (dobsondavanzo.com)
September 07, 2022 Short Url: https://ppsapta.org?04h8g0 Google+ Email Twitter
Industry Partner Webinar: EMG & MSK Ultrasound in the PT Private Practice: Challenges & Opportunities
Presented by APTA Private Practice Industry Partner Hands-On Diagnostics You can claim up to 0.1 CEUs for your participation (keep scrolling for details)!
EMG & MSK Ultrasound in the PT Private Practice: Challenges & Opportunities
Tuesday, August 30 | 7:00 PM ET
Bart McDonald, PT, MPT, ECS, FMSK
Dr. Dimitrios Kostopoulos, DPT, MD, PhD, DSc, ECS
Dr. Joe McGilvrey, DPT, ECS, FMSK
TAKE CE QUIZ
Empower your PT Private Practice with EMG & MSK Ultrasound: The diagnostic tools that will take your practice into the upper echelon of your industry.
Discover how EMG & MSK Ultrasound are improving the quality of care, helping therapists create more comprehensive treatment plans, changing the future of healthcare, and taking private practices to the next level. Learn how diagnostic testing can become a major revenue generator for your private practice and weigh the risks and benefits of implementation in your own practice.
Outline the clinical and financial benefits of EMG & MSK Ultrasound Testing in the PT private practice setting
Outline the steps needed to implement a new diagnostic tool in the practice setting, and predict the risks and benefits associated with it
Discuss state practice acts, CMS and other insurance regulations regarding EMG & MSK Ultrasound Testing in PT practice
Outline insurance billing codes for EMG & MSK Ultrasound and reimbursement patterns.
By the end of the session, participants will be able to evaluate the risks and benefits of EMG & MSK Ultrasound testing, decide if these services are appropriate for their own practice and further develop an implementation plan.
CEU Approval and Certification
APTA Private Practice, designates this educational webinar for up to 0.1 CEUs. Upon completion of watching the webinar and achieving a score of 70% or higher on the post-test, the participant will be eligible to claim the credit. Credit can be claimed until August 30, 2023.
To claim this free CEU credit, the learner will need to enter their name and email address to take the CEU exam. By doing so, the learner agrees to share their contact information with the sponsor.
APTA Private Practice online CEUs are accepted by the following physical therapy licensure boards, as allowed by the type of course requirements in state regulations: AL, AZ, CO, CT, DE, DC, FL, GA, HI, ID, IL, IN, IA, KS, KY, MI, MN, MS, MO, MT, NE, NH, NY, NC, ND, OR, PR, RI, SC, SD, TN, UT, VT, VA, WA, WV, WI, WY. Those in Oklahoma may apply individually for credit.
APTA Private Practice is accredited by the California Physical Therapy Association (Provider #CPTAAP-12) and the Texas Physical Therapy Association (Provider # 2008027TX). Consequently, all courses are accepted by California and Texas.
APTA Private Practice online courses are not automatically approved in AK, AR, LA, MD, NV, NM, NJ, OH, OK and PA, though individuals may be able to seek credit depending upon your state regulations.
APTA Private Practice issues CEUs/CCUs based on the following definitions: 1 CEU = 10 contact hours/10 CCUs. .1 CEU = 1 contact hour/1 CCU. 1 contact hour/1 CCU = 60 minutes of structured educational activity.
August 15, 2022 Short Url: https://ppsapta.org?vo4p6v Google+ Email Twitter
August Impact: "Friction" Now Online
August 01, 2022 Short Url: https://ppsapta.org?ky7xj1 Google+ Email Twitter
July Impact: "Ignite" Now Online
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June Impact: "Execute" Now Online
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May Impact: "Fortune" Now Online
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April Impact: "Chaos" Now Online
April 01, 2022 Short Url: https://ppsapta.org?ymav23 Google+ Email Twitter
COVID-19 State of Affairs (03/14/2022)
Open State of Affairs
The PPS COVID-19 State of Affairs document is updated weekly, with the newest information highlighted in red, to help you keep abreast of the most recent legislative and regulatory updates. Here are some important notes this week:
The Public Health Emergency (PHE) has been extended again and will be in place through mid-January 2022. PTs and PTAs are able to provide care to Medicare beneficiaries via telehealth only for the duration of the PHE. Whereas, PTs will be able to use virtual audio/visual technologies to achieve direct supervision until the end of the calendar year in which the PHE ends—through at least the end of 2022.
Medicare Coverage of Telehealth:
Physical therapists are eligible to provide and bill for Medicare services, as of 4/30/20 with a retroactive coverage date for claims beginning March 1, 2020. The Omnibus Federal Spending bill signed into law on March 11, 2022 includes a 151 day (~5 month) extension of Medicare coverage for real-time, face-to-face services provided by physical therapists and physical therapist assistants via telehealth after the Public Health Emergency (PHE) expires.
The current expiration date of the PHE is April 16, 2022. However, the administration has promised a 60 days "heads up" that they don't plan to renew the PHE; no such warning has been provided. Therefore it is anticipated that the PHE will be renewed in April and expire mid-July 2022.
Some waivers linked to the PHE expire when the PHE expires, some like the use of telehealth have been singled out for an extension, while others expire at the end of the year in which the PHE ends.
What We Don't Know:
When/If/How MACs will audit
Whether Congress will pursue another extension of current waivers or provide a permanent path to telehealth coverage for services provided by PTs and PTAs.
What You Can Do Now:
Advocacy Opportunity: Contact your Member of Congress and ask them to pass legislation to permanently enable physical therapists to provide physical therapy services to Medicare beneficiaries via telehealth.
Request that they cosponsor the Expanded Telehealth Access Act (S.3193/H.R.2168) which would permanently add physical therapists, physical therapist assistants, and other rehabilitation therapists to the statutory list of distant site providers that Medicare pays for telehealth.
Further ask that the text of the Expanded Telehealth Access Act be included in any long-term telehealth policy package that will be voted on by Congress.
March 14, 2022 Short Url: http://ppsapta.org?7kvgr6 Google+ Email Twitter
Industry Partner Webinar: Designing Effective Employee Compensation Models
Presented by PPS Industry Partner OnusOne You can claim up to 0.1 CEUs for your participation (keep scrolling for details)!
Designing Effective Employee Compensation Models
Tuesday, March 22 | 7:00 PM ET
Speaker: Jason Wambold, Co-Founder & Partner at OnusOne
Today's workforce carries an unprecedented debt load, demands higher salaries, and cites "work-life" balance as a top priority. Most employees expect pay to be commensurate with experience vs. production. Given the COVID pandemic and declining reimbursement, now is an ideal time to permanently alter the method by which we compensate rehab professionals. This session will present new/innovative concepts in compensation modeling.
Topics will include: shared risk/reward compensation models vs traditional bonus models, revenue-based vs volume based models, multiple plan tiers, best practice rollout strategy, compliance, and ethics.
Summarize evidence related to the effectiveness of traditional full salary plus bonus models
Identify the three most common barriers that prevent practices from effectively introducing alternative employee compensation models, and best practices to overcome those barriers
Describe/discuss the categorized strategic decisions which must be made in order to design effective alternative employee compensation models
Identify best practice as it relates to alternative employee compensation model rollout and implementation strategy
Summarize pertinent ethical considerations
The Private Practice Section, APTA, designates this educational webinar for up to 0.1 CEUs. Upon completion of watching the webinar and achieving a score of 70% or higher on the post-test, the participant will be eligible to claim the credit. Credit can be claimed until March 22, 2023.
Private Practice Section online CEUs are accepted by the following physical therapy licensure boards, as allowed by the type of course requirements in state regulations: AL, AZ, CO, CT, DE, DC, FL, GA, HI, ID, IL, IN, IA, KS, KY, MI, MN, MS, MO, MT, NE, NH, NY, NC, ND, OR, PR, RI, SC, SD, TN, UT, VT, VA, WA, WV, WI, WY. Those in Oklahoma may apply individually for credit.
The Private Practice Section is accredited by the California Physical Therapy Association (Provider #CPTAAP-12) and the Texas Physical Therapy Association (Provider # 2008027TX). Consequently, all courses are accepted by California and Texas.
PPS online courses are not automatically approved in AK, AR, LA, MD, NV, NM, NJ, OH, OK and PA, though individuals may be able to seek credit depending upon your state regulations.
PPS issues CEUs/CCUs based on the following definitions: 1 CEU = 10 contact hours/10 CCUs. .1 CEU = 1 contact hour/1 CCU. 1 contact hour/1 CCU = 60 minutes of structured educational activity.
March 07, 2022 Short Url: https://ppsapta.org?9rafqq Google+ Email Twitter
March Impact: "Elevate" Now Online
March 01, 2022 Short Url: https://ppsapta.org?u26c7c Google+ Email Twitter
February Impact: "Connection" Now Online
February 01, 2022 Short Url: http://ppsapta.org?9ror24 Google+ Email Twitter
No Surprises Act: Good Faith Estimate State of Affairs (2/04/2022)
UPDATED February 4, 2022
Open the No Surprises Act Good Faith Estimate State of Affairs here
The No Surprises Act (part of Public Law 116-260) requires certain disclosures related to surprise billing. In the implementation of the Act, CMS has promulgated regulations requiring that many providers including private practice PTs need to disclose a good faith estimate of costs of care to certain patients. Making this new requirement work in private practices has proven difficult, and PTs have raised many questions about how to comply. PPS has developed a new State of Affairs document available to keep you updated about the latest requirements as well as the work that PPS is doing to advocate on your behalf to decrease the administrative burden this new requirement imposes.
January 21, 2022 Short Url: http://ppsapta.org?1y1lge Google+ Email Twitter
January Impact: "Fearless" Now Online
January 03, 2022 Short Url: http://ppsapta.org?dck3e6 Google+ Email Twitter
November Impact: "Creating Your Plan" Now Online
November 01, 2021 Short Url: http://ppsapta.org?acodre Google+ Email Twitter
October Impact "Your Financial Future" Now Online
October 04, 2021 Short Url: http://ppsapta.org?3ta60h Google+ Email Twitter
September Toolkit: Physical Therapy is a Tree With Many Branches Now Online
September 01, 2021 Short Url: http://ppsapta.org?jvlm8e Google+ Email Twitter
September Impact: "Controlling Your Revenue" Now Online
August 30, 2021 Short Url: http://ppsapta.org?uepv50 Google+ Email Twitter
August Toolkit: Working in Physical Therapy Has Benefits That Extend Beyond the Clinic Now Online
August 03, 2021 Short Url: http://ppsapta.org?j0rlbl Google+ Email Twitter
August Impact: "Your Marketing Playbook" Now Online
August 01, 2021 Short Url: http://ppsapta.org?jz5izb Google+ Email Twitter
April Impact: Crafting Your Customer Experience
April 01, 2021 Short Url: http://ppsapta.org?qz2env Google+ Email Twitter
March Impact: "Staffing Your "A" Team" Now Online
March 01, 2021 Short Url: http://ppsapta.org?gfv5c8 Google+ Email Twitter
February Impact: "Leading Your Way" Now Online
February 03, 2021 Short Url: http://ppsapta.org?b0qzmn Google+ Email Twitter
New Marketing Toolkit Blog: #ChoosePT For Back Pain
May 03, 2019 Short Url: http://ppsapta.org?yd5wa0 Google+ Email Twitter
May Impact: "Beyond Brick & Mortar" Now Online
May 02, 2019 Short Url: http://ppsapta.org?7flb72 Google+ Email Twitter
April 2019 Legislative Update
April 29, 2019 Short Url: http://ppsapta.org?v5ilz7 Google+ Email Twitter
Nuts and Bolts of In-District Advocacy
April 17, 2019 Short Url: http://ppsapta.org?cbq42p Google+ Email Twitter
Letter to Congress: PPS and Alliance for Innovation in Medicare support the introduction of legislation to remove PTs from the In-Office Ancillary Services Exception (April 2019)
April 05, 2019 Short Url: http://ppsapta.org?ebzi9l Google+ Email Twitter
Letter to Congress: PPS recommendations to Congressional Telehealth Caucus (April 2019)
April 05, 2019 Short Url: http://ppsapta.org?5rzj52 Google+ Email Twitter
April Impact: "Swipe Right" Now Online
April 01, 2019 Short Url: http://ppsapta.org?nuhf0b Google+ Email Twitter
March 2019 Legislative Update
March 25, 2019 Short Url: http://ppsapta.org?45jlzl Google+ Email Twitter
February 2019 Legislative Update
February 25, 2019 Short Url: http://ppsapta.org?v2po4a Google+ Email Twitter
January 2019 Legislative Update
January 28, 2019 Short Url: http://ppsapta.org?ivo10m Google+ Email Twitter
New Legislative & Advocacy Priorities 116th Congress
January 18, 2019 Short Url: http://ppsapta.org?tdssxb Google+ Email Twitter
Advocacy Update: PPS Lobbyists response to the Affordable Care Act case being struck down
As I am sure you have heard on the news, on Friday 12/14, a federal judge in a lower court in TX filed a decision that declared the Affordable Care Act to be unconstitutional. His decision hinged on the fact that last December's tax bill the fine for not obtaining health insurance was reduced to zero. The decision held that since that fine has bee reduced to zero, that "tax" has been functionally removed from the law.
The reason this argument matters is that when the Supreme Court evaluated the constitutionality of the ACA previously, it determined that in fact the government did have the authority to compel Americans to have health insurance coverage, citing the Constitution granting Congress the authority to tax and spend. In yesterday's decision, the judge determined that since the "tax" has been reduced to zero, that authority has been relinquished and therefore the whole law must be struck (because of the argument that the taxing authority granted to Congress was the sole reason that it was able to compel Americans to have health insurance--and that the existence of the entire law hinged upon the taxing authority).
The ruling will not impact the 2019 health insurance plans sold through the exchange/marketplace of healthcare.gov or the state equivalents. It also won't immediately affect Americans' health coverage. The most immediate impact could be upon whether or not states will continue to consider expanding Medicaid at a time when the effort might be for naught.
An appeal is inevitable and already being discussed by a group of states led by California. They are likely to argue that the amount of the fine is immaterial, and that the $0 amount is not the equivalent of repealing the tax/fine and that the law still contains (and has the authority to impose) a fine. Because of the high stakes, and the political aspect of the law, the appeal process is likely to make its way to the US Supreme Court. This will take time.
If the US Supreme Court upholds the argument that the entire Congressional authority hinges on whether or not there is a tax/fine of more than $0 imposed upon those who do not have health insurance coverage, then the entire law will be invalidated. If that is the case, Americans will lose pre-existing condition protections, the ability of folks to stay on their parents' health insurance up to age 26 and other provisions of the ACA that have recently become popular on both sides of the aisle and the preservation of which has been a PPS legislative priority.
In a closely watched case, Judge Reed O'Connor of the Federal District Court in Fort Worth, Texas ruled that the Affordable Care Act's individual mandate, which Republicans zeroed out with their tax bill, "can no longer be sustained as an exercise of Congress's tax power." And the rest of the law cannot be separated from that provision and is therefore invalid, he wrote.
The New York Times: Texas Judge Strikes Down Obama's Affordable Care Act As Unconstitutional
A federal judge in Texas struck down the entire Affordable Care Act on Friday on the grounds that its mandate requiring people to buy health insurance is unconstitutional and the rest of the law cannot stand without it. The ruling was over a lawsuit filed this year by a group of Republican governors and state attorneys general. A group of intervening states led by Democrats promised to appeal the decision, which will most likely not have any immediate effect. (Goodnough and Pear, 12/14)
The Associated Press: Federal Judge Rules Health Care Overhaul Unconstitutional
In a 55-page opinion, U.S. District Judge Reed O'Connor ruled that last year's tax cut bill knocked the constitutional foundation from under "Obamacare" by eliminating a penalty for not having coverage. The rest of the law cannot be separated from that provision and is therefore invalid, he wrote. Supporters of the law immediately said they would appeal. "Today's misguided ruling will not deter us: our coalition will continue to fight in court for the health and wellbeing of all Americans," said California Attorney General Xavier Becerra, who is leading a coalition of states defending the ACA. (Alonso-Zaldivar, 12/14)
Politico: Judge Rules Obamacare Unconstitutional, Endangering Coverage For 20 Million
"In some ways, the question before the Court involves the intent of both the 2010 and 2017 Congresses," O'Connor wrote. "The former enacted the ACA. The latter sawed off the last leg it stood on." (Demko and Cancryn, 12/14)
Texas Tribune: Federal Judge Rules Obamacare Unconstitutional, Handing Texas An Early Win
In a ruling that could throw the nation's health care system into chaos, Fort Worth-based U.S. District Judge Reed O'Connor on Friday ruled that a major provision of the Affordable Care Act is unconstitutional — and that the rest of the landmark law must fall as well. In February, a Texas-led coalition of 20 states sued the federal government to end the health care law in its entirety, arguing that after Congress in December 2017 gutted one of its major provisions, the rest of the law was unconstitutional. (Platoff, 12/14)
Kaiser Health News: Judge Strikes Down ACA Putting Law In Legal Peril — Again
It is all but certain the case will become the third time the Supreme Court decides a constitutional question related to the ACA. In addition to upholding the law in 2012, the court rejected another challenge to the law in 2015. (Rovner, 12/14)
December 17, 2018 Short Url: http://ppsapta.org?eybq2g Google+ Email Twitter
Advocacy Update: PPS response to Anti-Kickback Statute
PPS' legislative priorities call for us to address and mitigate the negative effects associated with physician self-referral. In August, PPS filed formal comments in response to CMS' RFI on physician self-referral. On October 26th, PPS filed formal comments with the Department of Health and Human Services' Office of Inspector General urging both limits to the use of beneficiary incentives as well as transparency if providers are offering incentives to retain or induce patients to choose a particular provider. In both comments, PSS urged the prompt removal of PT from the IOASE and requesting increased accountability and transparency for referrals made utilizing the IOASE.
October 26, 2018 Short Url: http://ppsapta.org?nyiq6t Google+ Email Twitter
PPS comments on Department of Treasury's categorization of Physical Therapists for purposes of the Qualified Business Income Deduction, proposed rule
In December 2017, the Tax Cuts and Jobs Act provided for a permanent flat 21% tax rate for corporations. Under the law, qualified trades or businesses such as private practice physical therapists who pay their business taxes through their individual tax returns may be able to utilize a 20% tax deduction on qualified business income. The Department of Treasury has proposed to add physical therapists to the list of Specified Service Trades or Businesses (SSTB). If categorized as an SSTB, a physical therapist would NOT be eligible for the Code Section 199A deduction UNLESS their income was below $157,500 as an individual or $315,000 if married filing jointly. Once an income passes these initial thresholds, the deduction phases out for those whose incomes are between $315,000 and $415,000 for married taxpayers who file jointly and between $157,500 and $207,500 for single taxpayers. The deduction would not available for those whose income exceeds $415,000 (if married filing jointly).
PPS commented on the proposed rule, arguing that physical therapists should be treated as general qualified trades or businesses and not be added to the SSTB definition. Should the final rule not make the changes proposed by the Administration, a larger number of private practice section members would be able to utilize the 20% pass through tax deduction. Please consult your tax professional for advice as this tax break impacts the tax year beginning January 1, 2018.
October 02, 2018 Short Url: http://ppsapta.org?3k7eur Google+ Email Twitter
2018 Report: Impact of Physical Therapist Services on Low Back Pain Episodes of Care
September 17, 2018 Short Url: http://ppsapta.org?uzjuaw Google+ Email Twitter
Marketing, Finance, and Operations 101 Video Series
PPS has developed multiple educational video series to ensure that every PPS member has fundamental business management knowledge in Marketing, Finance, and Operations. The courses below can be accessed at anytime in the PPS Learning Center and are free to PPS members
In this 5-module, video series, learn how to define your target audience and develop a brand promise that engages customers. Discover marketing best practices, and tools to develop your marketing plan and accompanying budget. Finally, take a deep dive into three marketing strategies proven effective in marketing a physical therapy practice.
Module 1: Defining your target audience & developing a brand promise
Module 2: Developing a marketing plan and budget
Module 3: Referral source marketing
Module 4: Internal marketing
Module 5: Consumer/community outreach & marketing
REGISTER FOR MARKETING 101
OPERATIONS 101
Operations 101 provides an introduction to Key Performance Indicators and outlines the standardized definitions of recommended KPI's used to manage physical therapy practices. In modules two and three of this 3-part series, participants will learn how to use data and metrics to successfully manage practice operations, and will develop a strategy to create successful KPI performance.
Module 1: Introduction to Key Performance Indicators (KPI's)
Module 2: Using KPI's in your Physical Therapy Practice
Module 3: Management and Impact of KPI's on Your Practice
REGISTER FOR OPERATIONS 101
Finance 101 is a free, multi-module, video series that provides a solid foundation for those managing the finances of a physical therapy practice. The series was designed for non-financial people to gain improved understanding of bookkeeping methods, financial statements, and key terms requisite to successfully managing the finances of a physical therapy practice.
Module 1: Setting the Stage: Introducing key financial terms and concepts
Module 2: Finance for Non-Financial People
Module 3: Financial Statements
Module 4: Using Finance in Your Practice
Module 5: Bringing it All Together
Module 6: Real Life Questions
Module 7: How To and Tools
REGISTER FOR FINANCE 101
August 22, 2018 Short Url: http://ppsapta.org?3whtk1 Google+ Email Twitter
Recap: 2018 Presidential Address at the APTA House of Delegates
Three years after being elected President of the American Physical Therapy Association, Sharon L. Dunn, PT, PhD, stood before the House of Delegates and delivered an invigorating address to the body of electorates who first voted her into leadership back in 2015. At the heart of her message was a repeated and compelling charge to act as she emphasized that it is not enough to call out inadequacies in the system, or to idly tweet about embracing change or promoting inclusion. No, she reiterated that as a profession we must band together and work collaboratively to secure the future of our profession.
"The members of the American Physical Therapy Association were bold enough to dream of transforming society," she reminded, "Now we must deliver on that promise by following the most universal piece of advice we provide to our patients and clients: we must move!"
Memorable Quotes from President Dunn's speech are highlighted below:
SETTING A NEW TONE FOR THE FUTURE
"Most of us in this room wear those battle scars—and quite proudly. But where our past was shaped with sharp elbows, our future must be shaped with open arms. Where our origins situated our profession in recovery and the treatment of disorder, our future will be characterized by an increased role in sustaining health and proactively preventing disability and disease. And where previously we have demanded respect in part by staking out turf and occasionally pointing to the shortcomings of others, in our future we will demonstrate our value based on the irrefutable data of our own outcomes and in partnership with our colleagues across disciplines. The health care system and our patients will demand nothing less."
QUESTING OVER RESTING
"We should approach our future with enthusiasm, not dread, because our profession has never been satisfied with our position within the status quo. Our existence has been one of almost constant self-challenging evolution. Our progress has never been inevitable; it has always been hard-earned. So if we truly desire to not only see society transformed but to shape that transformation, we must not only weather the storm of health care disruption. Instead, as Dr Alan Jette urged in his 2012 Mary McMillan Lecture, we must face into the storm and choose questing over resting.
"But let's be clear: simply embracing change does not create change—and it doesn't make change easy."
WE MUST BE AGENTS, NOT CRITICS
"We cannot transform society through judgment. Our vision charges us not to stand at a distance and point our fingers at our nation's ills but instead to accept a personal responsibility to try to make a difference.
"At our core, we are healers, and as much as ever our country and our society need healing. It is not enough that we tweet about it in judgment. It is not enough that we adopt policies that articulate our principles. It is not enough that we sit idly by, in the comforts that were afforded by those who forged the path before us.
"No, our history tells us that when we stand together and act, we are a force."
LET NO ONE FEEL LIKE AN OUTSIDER
"To fulfill our mission, our community must vastly improve our diversity by being intentional about inclusion. We must prioritize listening and learning. We must enable the next generation of physical therapists and physical therapist assistants to transform our association, just as our association must transform society. Most of all we must get involved—with open arms and open minds—not only as a community, but as individuals."
OUR MOST VALUABLE ASSET: AN ENGAGED MEMBER
"When we formally charge APTA to act, or simply daydream about "what APTA should do...," we sometimes behave as if we're writing a check for someone else to cash. But as an individual membership association, we are charging ourselves-we are writing our own to-do list. APTA is not a distant factory that churns out ready-to-order physical therapy progress on a conveyor belt. It's a community of physical therapists, physical therapist assistants, and students. It's our community. We are APTA, and APTA is us. Our association's growth, commitment, and determination cannot outpace our own personal development."
FOLLOW OUR DREAMS WITH OUR OWN FOOTSTEPS
"If, like I do, you anticipate a day when physical therapists, like dentists, are part of our society's regular health routines, don't just point the way—raise a hand and accept a portion of responsibility to take us there.
"If, like I do, you believe that we gain strength not just with increased membership but with greater engagement, find a colleague who isn't as involved and demonstrate the spirit of inclusiveness our community requires.
"If, like I do, you believe that the power of our profession is boundless, that it can optimize movement and health in every corner of the world, then dare to look beyond traditional delivery and payment models; our patients and clients are likely just as frustrated by the limitations of the status quo as we are, and they are eager for the precise and personalized service we can provide.
"Above all else, if you believe, as I do, that our profession is more educated and skilled than ever before, that our potential is greater than it has ever been, then join me in demanding that we provide only the highest quality of care and that we are immediately accountable when the data suggest our patients and clients deserve something better."
APTA President Sharon L. Dunn, PT, PhD, Board-Certified Orthopaedic Clinical Specialist, was reelected to a second 3-year term, June 25, 2018. To read the full transcript of her 2018 address, or to watch the video, visit the APTA website.
June 28, 2018 Short Url: http://ppsapta.org?59uqqj Google+ Email Twitter
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A radical ecological activist who evaded capture for a decade pleaded guilty Thursday to conspiracy and arson for her role with an extremist ecoterrorism group believed responsible for setting 20 fires that burned $40 million of property across the West from 1996 to 2001.
Rebecca Jeanette Rubin, 40, a Canadian citizen, faces a federal prison term of five to 7½ years when she's sentenced in January, according to a plea agreement entered in U.S. District Court in Portland, Ore.
As part of the plea deal, Rubin, a former wildlife researcher, agreed to provide information about other members of the ecoterrorism group with which she has been connected. It's called "the Family," and according to the FBI, it was an even more extreme faction of two radical environmentalist groups, the Animal Liberation Front and the Earth Liberation Front.
But under the terms of the deal, which has been in negotiations for months, she won't be compelled to identify others involved in crimes committed by the Family — which federal authorities have branded the most wanted domestic terrorist group in the U.S.
Rubin surrendered last November at the Washington state border after a decade on the lam in Canada. Her attorney said that she tried to give herself up as early as 2009 but that he couldn't work out an acceptable arrangement with prosecutors.
Rubin was indicted in 2006 with three other Family members in a 1998 fire that caused $12 million at the Vail Mountain Ski Resort in Colorado. Two of the others, Chelsea Gerlach and Stanislas Meyerhoff, were sentenced to nine and 13 years in prison, respectively, in 2007. The third, Josephine Overaker, remains at large and is the subject of a $50,000 reward offered by the FBI.
The Family is accused of setting fires at 20 sites — including ski resorts, wild horse corrals, lumber mills and U.S. Forest Service offices — over five years beginning in 1996 in California, Colorado, Oregon, Washington and Wyoming.
In an extensive interview with NBC News when Rubin surrendered a year ago, Brent Smith, director of the Terrorism Research Center at University of Arkansas, said members were recruited after having proved themselves through extreme acts of environmental activism. They came from various walks of life: hippies, leftists, anarchists, some with criminal backgrounds, others with college degrees.
"These people got together, and first they would go to protests," Smith said. "There would be protests on campus where they protested destruction of trees to build a new building or something, and at these protests, kids become associated with each other."
Members would meet in small groups at what they called Book Clubs, where they would learn how to pick locks, monitor targets and make incendiary explosive devices to set fires.
"A lot of it was really spontaneous, which is very different from many of these true hard-core terrorist groups, where you see a lot of planning going on," Smith said.
In a detailed 2009 research paper on the Family, Smith identified the 1998 Vail fire — one of four that Rubin has been connected with — as the turning point that led to the Family's downfall.
After the highly publicized fire, members began questioning their tactics.
"During 2000, the group was inactive, and some of the members became disillusioned, believing that their tactics were not having the desired effects," Smith wrote.
"The ski resort at Vail, for example, was covered by insurance and was rebuilt in an even more grandiose fashion than before. In fact, all of the facilities they firebombed were rebuilt within a matter of months," he wrote.