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76ers' Joel Embiid Embraces 'Do a 180' Nickname After Viral 'Jeopardy' Video - Bleacher Report

Posted: 04 Mar 2020 10:18 PM PST

Philadelphia 76ers' Joel Embiid plays during an NBA basketball game against the Atlanta Hawks, Monday, Feb. 24, 2020, in Philadelphia. (AP Photo/Matt Slocum)
Matt Slocum/Associated Press

Philadelphia 76ers big man Joel Embiid rarely misses an opportunity to go viral.

The three-time All-Star was the subject of a Jeopardy! question Tuesday when Alex Trebek asked the contestants what famous nickname Embiid trademarked when the 76ers were looking to turn things around through plenty of losses and high draft picks early in his career.

Even casual NBA fans would know it was "The Process," but contestant Paul was just a bit off with his guess of "do a 180."

Embiid responded with an amusing highlight of him turning the ball over as a young player as he did a 180 jump behind the backboard and even changing his Twitter name.

It's time for 76ers fans to start trusting the 180.

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Brodie's goal late in OT lifts Flames past Blue Jackets 3-2 - USA TODAY

Posted: 04 Mar 2020 10:08 PM PST

AP

Updated 12:47 AM EST Mar 5, 2020

CALGARY, Alberta (AP) — T.J. Brodie scored with 11 seconds remaining in overtime as the Calgary Flames rallied from a two-goal deficit and beat the Columbus Blue Jackets 3-2 on Wednesday night.

Blue Jackets goaltender Joonas Korpisalo had lost his stick and was unable to get a glove on Brodie's wrist shot from the high slot.

Elias Lindholm and Matthew Tkachuk also scored for Calgary. Cam Talbot made 20 saves and improved to 8-3-1 since Jan. 1.

Calgary (35-26-7) continued to cling to third place in the Pacific Division, just ahead of five teams jockeying for Western Conference wild-card berths.

"We're desperate right now," Flames captain Mark Giordano said. "We know where we are in the standings. Every night is huge. Your nights off, you see teams winning around you. When you play you've got to make it count.

"The effort was there all night. Sometimes it doesn't look as good from up top because the other team is doing a pretty good job out there too.

Devin Shore and Gustav Nyquist scored first-period goals for Columbus. Korpisalo stopped 34 shots.

At the start of a five-game homestand, the Flames improved their record at the Saddledome to 14-13-4.

"I think there's been such an emphasis being talked about how we can't play at home ... how we're terrible at home," Calgary head coach Geoff Ward said. "That's all we've been hearing around home for awhile now.

"Probably the fact we were able to break through and do what we did tonight, it can be more of a confidence builder for us than maybe in a different situation when the emphasis wasn't so much on how bad we are at home."

Lindholm kicked off Calgary's comeback at 11:14 of the third period.

With Talbot pulled for an extra attacker, Tkachuk scored a tying goal at 18:17.

The injury-riddled Blue Jackets have forwards Cam Atkinson (ankle) and Brandon Dubinsky (wrist), defensemen Seth Jones (ankle) and Dean Kukan (knee), winger Josh Anderson (shoulder) and rookie centre Alex Texier (back) on injured reserve.

Forwards Oliver Bjorkstrand (ankle) and Nathan Gerbe (groin) are also sidelined, while goaltender Matiss Merzlikins is day-to-day with an upper-body injury.

Columbus defenseman Ryan Murray played just his second game Wednesday since breaking his hand in December.

"Don't even start talking about injuries," Blue Jackets head coach John Tortorella said. "This has nothing to do with it.

"We've been doing this for three months and we have found a way to win games. We need to continue to stay with it.

"We need to get some guys that I think can bring more offense to do that more consistently without sacrificing some of the things we need to do above the puck."

With Talbot pulled for an extra attacker, Flames captain Mark Giordano threaded a shot through traffic for Tkachuk to re-direct home.

Lindholm pulled the hosts within a goal midway through the third.

His linemate Andrew Mangiapane harassed defenseman Zach Werenski behind the Columbus net and forced a turnover for Lindholm to exploit.

The Flames fanned on scoring opportunities in the second period.

Calgary's Tobias Rieder missed the net on a breakaway, Korpisalo stoned Mikael Backlund from close range and Lindholm couldn't corral a bouncing rebound on a shot by Johnny Gaudreau.

Columbus turned Calgary's neutral zone turnover into an odd-man rush with Nyquist converting a feed from Nick Foligno at 11:20 of the opening period.

Shore scored his first goal as a Blue Jacket. He was acquired from Anaheim at the trade deadline on Feb. 24. The winger squeezed a wrist shot from the face-off circle under Talbot's right arm at 8:56.

Tkachuk was hobbled blocking a shot at the four-minute mark. He remained in the game, but skated uncomfortably at times.

"It's important for us to come and take care of business here at home, which we haven't done as of late, but hopefully we can turn it around," Tkachuk said. "This is a big month of a lot of home games."

NOTES: Flames forward Derek Ryan skated in his 300th career NHL game. ... Defensemen Erik Gustafsson and Derek Forbort played their first home games as Flames since being acquired at the trade deadline.

UP NEXT

Blue Jackets: At Edmonton Oilers on Saturday.

Flames: Host Arizona Coyotes on Friday.

___

More AP NHL: https://apnews.com/NHL and https://twitter.com/AP_Sports

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東宝スタジオ内に「バンダイナムコ研究所 東宝スタジオラボ」を新設 - PR TIMES

Posted: 04 Mar 2020 09:44 PM PST

バンダイナムコ研究所は「Creating Entertainment Innovation!（革新的なエンターテインメントを創造する）」というミッションのもと、最先端技術における研究・検証・開発や、ゲーム制作で培われたノウハウを活用した新たなコンテンツの研究開発を手掛けています。昨今、AIやAR（拡張現実）・VR（仮想現実）・MR（複合現実）の技術開発が加速するなか、エンターテインメントをはじめ、さまざまな分野において「人を笑顔にする」可能性が広がりつつあり、迅速に研究開発を進めるためにはプロトタイピング環境が必要不可欠になりました。東宝スタジオは日本最大級のスタジオで、映像・音声ともに最新鋭の技術を搭載した施設を兼ね備えているほか、映像における膨大な知識と経験豊富なクリエイターが多く集まっていることから、さまざまなチャレンジが期待できます。
今後は、バンダイナムコ研究所の試作実験場として最大限活用していきますのでご期待ください。

【概要】
名称： バンダイナムコ研究所 東宝スタジオラボ
住所： 〒157-0066　東京都世田谷区成城1-4-1
東宝スタジオ プロダクションセンター内　SR407、SR408
開所日 ：2020年2月7日

バンダイナムコ研究所　東宝スタジオラボバンダイナムコ研究所　東宝スタジオラボ

バンダイナムコ研究所　東宝スタジオラボバンダイナムコ研究所　東宝スタジオラボ


 


【東宝スタジオ　概要】
名称：東宝スタジオ
設立：1932年
敷地面積：約78,000㎡（約23,000坪）
撮影ステージ数： 10棟
ポストプロダクションセンター：2棟
 ※「バンダイナムコ研究所 東宝スタジオラボ」は東宝スタジオ内のプロダクションセンターにあります。

東宝スタジオサービス東宝スタジオサービス

■バンダイナムコ研究所　　代表取締役社長　中谷 始
バンダイナムコ研究所は、最先端技術の研究開発を起点に「エンターテインメントの新しい価値を創出」するエンターテインメント イノベーション集団です。そのために、世界中のイノベーターや研究者、企業と積極的に共創し、一緒に新しいエンターテインメントを創出していきたいと強く願っています。このたび、東宝スタジオ様のご厚意で、素晴らしい環境をお借りできることになりました。今後は「バンダイナムコ研究所 東宝スタジオラボ」から、世界に向けて革新的なエンターテインメントを発信していきます。

■東宝スタジオ　　東宝株式会社　取締役　山下 誠 様
東宝スタジオは1932年、前身の写真化学研究所（PCL）として設立以降、80年以上もの間、映画をはじめとした様々なエンターテインメントを、世界に向けて発信し続けてまいりました。2004年には「スタジオ改造計画」として大規模な再開発に着手。足かけ8年をかけた大改造により、東宝スタジオは、プリプロダクションからポストプロダクションまでの一貫した生産ラインを提供できる近代スタジオに生まれ変わりました。このたび、バンダイナムコ研究所様が新たに東宝スタジオに加わって頂けることを心から歓迎いたします。
そして、これからもより一層新しいエンターテインメントの世界が広がることを、楽しみにしております。

以上

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Viral evolution identifies a regulatory interface between paramyxovirus polymerase complex and nucleocapsid that controls replication dynamics - Science Advances

Posted: 04 Mar 2020 09:18 PM PST

Abstract

Paramyxoviruses are negative-polarity RNA viruses of major clinical importance. The dynamic interaction of the RNA-dependent RNA polymerase (RdRP) complex with the encapsidated RNA genome is mechanistically and structurally poorly understood. Having generated recombinant measles (MeV) and canine distemper (CDV) viruses with truncated nucleocapsid (N) protein showing defects in replication kinetics, we have applied a viral evolution approach to the problem. Passaging of recombinants resulted in long-range compensatory mutations that restored RdRP bioactivity in minigenome assays and efficient replication of engineered viruses. Compensatory mutations clustered at an electronically compatible acidic loop in N-core and a basic face of the phosphoprotein X domain (P-XD). Co-affinity precipitations, biolayer interferometry, and molecular docking revealed an electrostatic-driven transiently forming interface between these domains. The compensatory mutations reduced electrostatic compatibility of these microdomains and lowered coprecipitation efficiency, consistent with a molecular checkpoint function that regulates paramyxovirus polymerase mobility through modulation of conformational stability of the P-XD assembly.

INTRODUCTION

The order Mononegavirales encompasses nonsegmented RNA virus families with negative-polarity genomes. Several of these families contain major human and animal pathogens, such as Ebola virus, rabies virus, measles virus (MeV), and respiratory syncytial virus (RSV) (1). MeV and several additional members of the paramyxovirus family such as mumps virus (MuV) and the parainfluenza viruses are the etiologic agents of some of the most severe pediatric viral diseases.

Although the family is large and expanding, central features of genome architecture and RNA-dependent RNA polymerase (RdRP) complex organization are well conserved (2). The viral genomes are N-encapsidated, and the resulting ribonucleoprotein (RNP) complexes are organized in a helical configuration. The viral polymerase (L) protein contains all enzymatic activities necessary for RNA synthesis, which includes phosphodiester bond formation, polyadenylation, mRNA capping, and cap methylation, and is recruited to the RNPs by the P protein (Fig. 1A). P is a homotetramer with a modular organization in which short, structured functional domains are interspersed by large structurally disordered segments. Two interacting domains between the P and N proteins have been identified: (i) a C-terminal three-helix bundle X domain (P-XD) that interacts with a molecular recognition element (MoRE) located in the large, unstructured C-terminal N-tail that is present in morbilliviruses, henipaviruses, and potentially respiroviruses, and (ii) an N-terminal molecular response element in P that has been shown to interact with the structured core domain of free N monomers, preventing premature N multimerization and preserving an RNA-free state of N (3–5).

Fig. 1 Recovery of recCDVs with tail-truncated N proteins.

(A) Schematic of the RdRP complex showing N (blue, cyan, and green) and N-tail binding P-XD (green) through N-MoRE (orange). L protein docks to the P C terminus (14, 26). (B) CDV N is organized as N-core (amino acids 1 to 391) and N-tail (amino acids 392 to 523) domains. N-tail contains modules of conserved residues: boxes 1 to 3 (yellow, orange, and red). Box 2 harbors N-MoRE. The [425–479] junction is represented below. (C) Minigenome reporter assays to determine RdRP bioactivity. Relative luciferase units were normalized to the peak activity in the presence of full-length N (n = 12) [nonlinear regression using a log(normal) model]. Error bars: Geometric SD. Two-way analysis of variance (ANOVA) with Sidak's post hoc test. ns, not statistically significant. (D) Enumeration of progeny syncytia after transfer of initial infectious centers. Mean (n = 9) represented as black bar. Kruskal-Wallis test with post hoc Dunn's test. (E) Representative microphotographs (×100 magnification) of recCDV CPE at 35 hours post-infection (h.p.i.). (F) Mean syncytium extension of three independently recovered recombinant viruses (14, 22, and 35 hours post-infection). (G) Substitutions reported for N and P ORFs of recombinant CDV encoding full-length (n = 3) or Δ[425–479] (n = 10) N after 10 passages. (−): no substitution.

Mononegavirales nucleocapsid high-resolution structures are available for a number of representative viruses, including MeV (6, 7), human parainfluenza virus 5 (HPIV-5) (8), and RSV (9). Unstructured N-tail–like domains were found to be conserved among the paramyxoviruses and filoviruses but absent from pneumovirus and rhabdovirus capsids. Furthermore, high-resolution structures of MoRE assemblies with P-XD were solved for MeV and Hendra virus (HeV), which showed a four-helix arrangement consisting of three P-XD–derived helices forming a triangular prism that interacts with a single MoRE-derived α-helix. Although MoRE binding to P-XD is not required for initial recruitment of the polymerase to the RNP template (10), N-MoRE:P-XD interactions are important to prevent premature RdRP termination and correct negotiation of intergenic junctions (11, 12).

The KD (dissociation constant) of the isolated MeV, HeV, and NiV (Nipah virus) N-MoRE interaction with P-XD is 2 to 20 μM (13). However, the tetrameric nature of P and the high density of N-tails in close proximity to the polymerase complex loaded onto the helical RNP template generate high avidity. It therefore remains elusive how advance of MeV—and in fact any mononegavirus—RdRP complexes along the encapsidated template is achieved and regulated, which requires repetitive dissolution and reformation of P interaction not only with N but also with P-XD and L, as recently shown (14). It was suggested that the unstructured central MeV N-tail section located upstream of MoRE may contribute to MoRE separation from P-XD (13). However, we have successfully relocated MoRE into N-core in recombinant, replication-competent MeV (12) and removed all of the central N-tail section (approximately 60 amino acids) without negatively affecting RdRP bioactivity in minigenome assays (15). We also successfully recovered closely related canine distemper virus (CDV) recombinants with corresponding N-tail truncations, which replicated efficiently in cell culture and remained partially pathogenic in the ferret model of CDV infection (15). These findings demonstrated that paramyxovirus polymerase bioactivity does not depend on the physical presence of the unstructured N-tail section. However, genetic evaluation of the N-tail truncated CDV revealed that robust virus growth coincided with the appearance of a mutation at residue 156 in N-core (15), implying that the deleted N-tail section may function as a long-range regulator of RdRP activity.

In the present study, we implemented an in vitro virus evolution approach to elucidate this mechanistic role of unstructured N-tail domains in virus replication. Having identified and validated a panel of compensatory mutations that emerged independently in N-core or P-XD in distinct selection lineages, we found that experimentally confirmed substitutions restoring efficient virus replication clustered in electronically compatible N-core and P-XD microdomains. Direct interactions between these domains were tested biochemically using purified proteins and through biolayer interferometry, confirming the identification of a previously unidentified, intermediate-affinity interface between RNP-embedded N-core and P-XD that is critical for RdRP bioactivity. The spatial location and electronic properties of the microdomains are conserved across diverse paramyxoviruses. Our results suggest that unstructured mononegavirus N-tail domains evolved as major checkpoints for viral polymerase dynamics.

RESULTS

The N-tail–modified CDV that we previously recovered harbored a large internal tail truncation spanning residues 425 to 479 (Fig. 1B). This virus remained partially pathogenic in the ferret animal model of CDV infection (15). Deep sequencing revealed two point mutations in the N-core domain and at the origin of N-tail, E156Q and A410D, respectively, raising the question of whether these changes were instrumental for the efficient growth of the recombinant through a compensatory effect.

Fitness penalty of N-tail truncation is compensated through point mutations in N or P

To test this hypothesis, we first validated bioactivity of the original tail-truncated N lacking these mutations in plasmid-based minigenome assays (Fig. 1C). Because RdRP bioactivity is critically affected by the relative ratio of nucleocapsid and polymerase complexes, we generated activity profiles of mutant and standard N over a wide ratio range. In the presence of standard N, RdRP activity adhered to a log-normal model with increasing relative P-L amounts, reaching a peak at a 4.9:1 (N:L-P) ratio. The tail-truncated N mutant likewise followed a ratio-dependent activity optimum curve, but peak signal was reached at a lower 3:1 (N:L-P) ratio and peak height was reduced to 43% compared to standard N. Activity profiling thus confirmed that the tail-truncated N is bioactive but revealed a statistically significant activity penalty.

To explore whether distinct avenues to compensate tail truncations are available to the protein complex, we sought to expand the portfolio of compensatory mutation candidates through multiple independent virus recovery transfections from cloned complementary DNA (cDNA), containing either standard N (recCDV N wt; three independent transfections) (16) or tail-truncated N [recCDV NΔ[425–479]; 11 independent transfections (15)]. We transferred three individual infectious centers of each recovery transfection reaction to CDV-permissive Vero cells stably expressing canine SLAM (Vero-cSLAM) when multinucleated giant cells or syncytia, which represent the predominant cytopathic effect (CPE) associated with CDV infection, became detectable microscopically. Within 48 hours of transfer, cultures having received standard recCDV N wt infectious centers showed extensive CPE, producing a mean of 52 progeny syncytia each (Fig. 1D). By comparison, recovery of recCDV NΔ[425–479] was likewise successful, but initial virus spread was drastically reduced with a mean of only 0.89 (0 to 4) syncytia observed 48 hours after transfer. To assess virus growth kinetics at this very early stage after recovery [passage 1 (p1)], we quantified the enlargement of this inaugural round of progeny syncytia. Standard recCDV N wt replicated without appreciable time delay after recovery, reflected by rapid expansion of syncytia (Fig. 1, E and F). In contrast, recCDV NΔ[425–479] followed a biphasic initial replication pattern. First, infectious centers expanded slowly, requiring passaging of infected cells to maintain cultures in the most permissive exponential cell growth phase. After approximately 60 hours, however, syncytia growth rates increased drastically to a level equivalent to that of standard recCDV N wt. All subsequent infections of cells with these recCDV NΔ[425–479] populations resulted in rapid, recCDV N wt–like CPE formation.

Having subjected 10 independently recovered recCDV NΔ[425–479] populations to 10 consecutive passages each on Vero-cSLAM cells, we Sanger-sequenced the viral N and P genes (Fig. 1G). Seven of these p10 virus populations contained allele-dominant point mutations in N, which were located in N-tail in the two candidates and in N-core in the other five. Two virus populations featured mutations exclusively in the P protein, and only one contained changes in both N and P. Whenever mutations in P emerged, at least one substitution was located in P-XD. By comparison, none of the three independently recovered and passaged standard recCDV N wt populations acquired changes in the N or P protein.

The biphasic initial replication kinetic of all newly recovered recCDV NΔ[425–479] and the identification of allele-dominant substitutions in N and/or P exclusively in these populations are consistent with rapid in cellula evolution of the N-tail truncated recCDV after initial recovery. These observations suggest that the individual mutations restore viral fitness through compensatory effects. Most of the substitutions were located outside of N-tail—only one candidate contained a change (R424S) in immediate proximity of the truncation site—indicating that compensation is achieved mechanistically through long-range effects.

Compensatory mutation candidates cluster in distinct N-core and P-XD microdomains

Sequence alignment of different morbillivirus N proteins revealed that the substitutions affect residues that are well conserved within the paramyxovirus genus (fig. S1). To test for compensatory effects, we rebuilt all candidate mutations in the context of both N wt and NΔ[425–479] and determined activity profiles over the broad P-L to N ratio in minigenome assays (three selected candidates with mutations in different areas are shown in Fig. 2A; all others are shown in figs. S2 and S3A). Comparing informative peak RdRP activities, we identified three distinct mutations in N and five in P that statistically significantly restored bioactivity of NΔ[425–479] in the minigenome assays (Fig. 2, B and C), including the N-E156Q A410D double mutation that had first emerged in our previous study (15). CDV strains 5804PeH (used for adaptation) and Onderstepoort (the basis for the minigenome system) contain a natural A/T allele variation of N residue 410. However, insertion of a T410A substitution in the Onderstepoort minigenome system did not affect polymerase activity, indicating that this variation has no functional impact (fig. S3B).

Fig. 2 Identification of specific mutations compensating for N-tail truncation.

(A) Minigenome activity profiles of full-length (blue) and tail-truncated (red) N. Normalized activities of selected mutations in full-length (blue line and symbols) and tail-truncated (red line and symbols) N background (n = 3) are overlaid. A natural allele A/T allele variation exists at residue 410 between CDV strains 5804PeH and Onderstepoort. Statistical analysis and regression modeling as in Fig. 1C. (B and C) Peak minigenome activities after rebuilding of candidate substitutions, analyzed as in (A). Each set of peak activity in the context of either N or Δ[425–479] was compared with one-way ANOVA with Dunnett's multiple comparisons post hoc test. Mutations associated with statistically significant polymerase activity differences in either N background or showing the greatest increase in the presence of tail-truncated N are highlighted with yellow shading. (D) Localization of compensatory mutation candidates in N-core. Surface representation (left) and ribbon model enlargements (right) of mutation sites are shown [Protein Data Bank (PDB): 4UFT]. Substituted side chains as sticks, color-coded by positive (green), neutral (blue), or negative (red) effect on RdRP activity in the distinct N-tail background. (E) Localization of compensatory mutation candidates in P-XD. Ribbon models of P-XD (PDB: 1T6O) are shown, color coding as in (D).

When we located the different N-core substitutions in a homology model of CDV nucleocapsid that was generated on the basis of the cryo–electron microscopy reconstruction reported for MeV (6), D151E, D154G, E156Q [associated with statistically unchanged or significantly higher minigenome activity in the NΔ[425–479] background], and F209C were predicted to reside in close proximity to each other in a defined microdomain located on the outer surface of the RNP helix (Fig. 2D). The remaining mutation, E103K, was posited between consecutive rungs of the helical RNP assembly, possibly in proximity to the origin of N-tail protruding from the RNP at the intersections between neighboring rings.

While most of N-tail is intrinsically disordered, three substitutions appeared one each in the three conserved tail subdomains: box1 (S415P), box2 (L503F), and box3 (R519G) (Fig. 1A). Minigenome activity testing revealed three distinct phenotypes associated with these changes: S415P statistically significantly increased RdRP bioactivity in both truncated and full-length N-tail background (113 and 165%, respectively), L503F had no major effect on bioactivity (66 and 72%, respectively), and R519G caused activity losses in either N background (12 and 32%, respectively) (Fig. 2B). These findings underscore the importance of box1 residues in regulating polymerase bioactivity.

Although morbillivirus P proteins are more sequence diverse than the N proteins, P mutations A465V, R469G, H479N, and K486N are all located in the conserved C-terminal P-XD (Fig. 2E). All of these substitutions statistically significantly boosted RdRP bioactivity in minigenome assays in the context of tail-truncated NΔ[425–479], while combination with standard N wt had either no statistically significant effect or reduced (R469G) bioactivity (Fig. 2C). A crystal structure has been released for MeV P-XD, revealing folding into a triangular prism conformation (17). When located in a corresponding CDV P-XD model, we found all four mutations to populate a single side of the prism created by α-helices 1 and 2 (Fig. 2E). This α1α2 face is distinct from the known P-XD interfaces α2α3 with the N-MoRE domain (18, 19) and α1α3 with the L protein (14) and has not previously been implicated in engaging in protein-protein interactions. Only one P protein mutation, P42L, was located outside of P-XD in a more variable zone near the N terminus of the protein.

Recombinant, tail-truncated CDVs with candidate compensation mutations regain full fitness and are genetically stable

On the basis of the performance in minigenome activity assays and location in diverse areas of the N and P proteins, we selected the N-E156Q A410D double mutation, N-S415P, and P-R469G in P-XD for reconstruction in the context of standard and N-tail–truncated recombinant CDVs (Fig. 3A) and determined recovery profiles and growth kinetics. All recCDVs carrying either of these mutations in the NΔ[425–479] background were recovered as efficient as standard recCDV, but each of the substitutions negatively affected virus recovery when combined with N wt (Fig. 3B). Initial replication kinetics of the three recCDV NΔ[425–479] with the reconstructed substitutions were likewise indistinguishable from that of standard recCDV (Fig. 3C), while recCDVs with full-length N showed initial growth delays (N-E156Q/A410D mutation) or a severe and lasting growth block (N-S415P).

Fig. 3 Recovery of recCDV with confirmed compensatory mutations.

(A) Schematic of recCDV genome organization [red star: N-E156Q + A410D (CDV 5804PeH background); green star: N-S415P; blue star: P-R469G; mutant color coding is maintained in all subpanels of this figure]. (B) Enumeration of progeny syncytia after transfer of initial infectious centers after recovery. (C) Mean syncytium extension of three independently recovered recombinants each, analyzed at 14, 22, and 35 hours post-infection. (D) Genetic stability of recCDV with full-length (n = 3) or Δ[425–479] (n = 10) N and engineered substitutions as specified after five passages. N and P genes were analyzed. (E) Multicycle growth profiles of recCDV as specified. Symbols represent mean viral titers (n = 3), and error bars denote SD to the mean. Two-way ANOVA with Dunnett's multiple comparisons post hoc test.

To assess genetic stability of the different CDVs, we subjected all recombinants to five consecutive passages in three independent replicates each, followed by sequence analysis of the N and P open reading frames (ORFs) (Fig. 3D). While most candidates were genetically stable, the N-S415P substitution triggered, in all cases, additional substitutions when present in recCDVs with full-length N. One replicate of recCDV with N-tail truncation and N-S415P developed a change in P (P-L148R), although this substitution was not allele dominant after five passages (fig. S4). Multicycle viral growth curves confirmed that standard recCDV-like replication rates and peak titers were restored in all three recCDV NΔ[425–479] populations carrying the reconstructed mutations (Fig. 3E). Maximal progeny titers of recCDV NΔ[425–479] N-S415P slightly exceeded those of recCDV N wt.

These observations demonstrate that the tested mutations in N and P fully compensate for the NΔ[425–479] tail truncation under physiologically relevant conditions of virus replication in cultured cells, establishing a functional link between the microdomain identified on N-core and the α1α2 face of P-XD.

Compensatory mutations are located in microdomains with complementary electronic properties

Inspection of the nature of the compensatory substitutions improving RdRP activity in the minigenome assays revealed that the majority affected negatively charged residues in N-core (D151, D154, and E156) and positively charged amino acids in P-XD (R469, H479, and K486) (Fig. 4, A to D). To directly probe the importance of charge in these microdomains for bioactivity, we generated a series of charge-neutralization and/or charge-reversal mutations covering both areas and assessed the effect on RdRP bioactivity in the context of standard and tail-truncated N (Fig. 4, E to H, and figs. S5 and S6). All charge reversals in the acidic loop in N-core abrogated or statistically significantly reduced polymerase activity in the context of both full-length and tail-truncated N (illustrated for residues D156 and D158 in Fig. 4E, summarized for all substitutions tested in Fig. 4F). Charge neutralization was better tolerated, returning peak activities of 52 and 39% residual activities for N-E148N in the context of standard and tail-truncated N proteins, respectively, and 134 and 20% for D158N. Both E161N and E208N eliminated polymerase activity.

Fig. 4 Electronic properties of compensatory microdomains in N-core and P-XD.

(A to D) Ribbon (A and C) and electrostatic surface (B and D) representations of compensatory microdomains in CDV N-core (A and B) and P-XD (C and D). Residues subjected to mutagenesis are labeled, and negative (red) and positive (blue) charges are indicated. (E to H) Peak minigenome activities in the distinct N-tail backgrounds after charge-reversal or charge-neutralization mutagenesis as specified in N (E and F) or P (G and H). Statistical analysis and regression modeling as in Fig. 2 (B and C). One-way ANOVA with Holm-Sidak's multiple comparisons post hoc test.

Altering the positively charged α1α2 P-XD face of the P-XD helix bundle was more agreeable with polymerase bioactivity overall (Fig. 4, G and H). The activity was unchanged by mutation P-R469E and drastically increased by P-R472E charge reversal in the context of tail-truncated NΔ[425–479] (163% of reference activity; Fig. 4H). Noteworthy, the P-R472E substitution had no effect on RdRP activity in the context of full-length N. Double and triple combinations of charge-reversal mutations moderately to drastically reduced polymerase activity in the context of both standard and truncated N. Whereas introduction of either a positive or negative charge at position 465 reduced activity with tail-truncated N, only a negative charge had a clear impact on full-length N (~51%) (Fig. 4H and fig. S6).

These results reveal that maintaining a high local density of negative charge in the N-core microdomain is required for polymerase functionality. Greater resilience of the α1α2 P-XD face to charge reversal suggests more redundancy in this system. However, the differential effect of P-R472E and A465D or A465R on RdRP bioactivity in the context of NΔ[425–479] versus full-length N emphasizes the functional link between the presence of the unstructured central N-tail section and the α1α2 face of P-XD.

Purified P-XD:N-MoRE complex specifically interacts with N-core

To biochemically test whether the acidic N loop and P-XD microdomains defined by the compensatory mutations interact with each other, we purified glutathione S-transferase (GST)–tagged CDV P-XD and P-XD:N-MoRE complexes after prokaryotic expression (Fig. 5A), the latter synthesized as single polypeptides as described for MeV (18). Independently, we purified hexahistidine-tagged N-core fragments truncated after tail residue 419, which reportedly prevents compaction of the corresponding nucleocapsids (20). Two mutated versions of these proteins, P-R469E and N-E156K in P-XD and N-core, respectively, were generated and purified in parallel to determine specificity of candidate interactions.

Fig. 5 Biochemical interaction of compensatory microdomains in N-core and P-XD.

(A) Top: Ribbon representation of P-XD (pink)/N-MoRE (orange) (PDB: 1T6O). Bottom: Triangular prism configuration of P-XD. (B) Affinity coprecipitation with Glutathione Sepharose beads. Coomassie blue–stained gels (left) and anti-CDV N immunoblots (right) of eluted protein fractions after SDS–polyacrylamide gel electrophoresis are shown. *Copurified cellular contaminants. (C) Densitometry of coprecipitations. Values denote precipitation efficiencies relative to input material (n ≥ 3 ± SD). One-way ANOVA with Holm-Sidak's multiple comparisons post hoc test. (D) Biolayer interferometry showing association and dissociation of N[1–419] ± E156K substitution with biosensors loaded with indicated GST fusions. (E) Affinity coprecipitation as in (B) with natural P-XD compensatory mutations A465V and A469G and engineered A465R. Coomassie blue–stained gels. (F) Densitometry of coprecipitations as in (C) (n = 5 ± SD). One-way ANOVA with Holm-Sidak's multiple comparisons post hoc test. (G) Phenotypic effects of compensatory and engineered mutations at P positions 465 and 469. (H) Molecular docking of P-XD:N-MoRE complexes (PDB: 1T6O) into N-core (PDB: 4UFT). Top: Surface of N protomers (blue, purple, and green) and P-XD (pink) with N-MoRE (orange). Bottom: Predicted contacts between P-XD and N-core acidic loop and N-MoRE with N-core loop (residues 133 to 142; yellow). Right: Docking scores and PRODIGY predictions (36).

With an electrophoretically authenticated set of purified proteins at hand (fig. S7), we analyzed the direct interaction of N-core and P-XD:N-MoRE through affinity coprecipitation followed by gel fractionation (Fig. 5B). Approximately 25% (±17%) of the N[1–419] input material coprecipitated with P-XD:N-MoRE, demonstrating efficient interaction of the proteins (Fig. 5C). In contrast, free P-XD was unable to bind to N[1–419]. GST alone also did not interact with N[1–419], and introduction of the E156K charge-reversal mutation into N-core abrogated binding. For orthogonal validation of these coprecipitation results, we used biolayer interferometry for label-free quantitative assessment of protein binding using GST-specific biosensors coated with the different GST-tagged P-XD polypeptides or, for reference, GST alone. All sensors were tested against standard or mutant N[1–419] polypeptides at a concentration range of 0.5 to 4 μM (Fig. 5D). Concentration-dependent interactions in the micromolar range were detected between P-XD:N-MoRE complexes and standard N[1–419], although precise kinetics could not reasonably be extracted because of the large number of binding sites present in the multimeric N assemblies and the GST-fusion protein dimers. Binding of free P-XD lacking MoRE to N[1–419] was also detected, albeit substantially weaker. Each of the engineered point mutations in N-core and P-XD abolished binding, and interaction of N[1–419] with biosensors coated with free GST remained negligible, confirming specificity of the assay. This panel of biochemical assays therefore revealed consistently that P-XD:N-MoRE complexes interact specifically with N-core and implicates positively charged residues located in the α1α2 face of P-XD prism as instrumental for efficient binding.

Having validated the co-affinity precipitation assay, we explored the effect of specific P-XD mutations that emerged from viral evolution, A465V and A469G, on the P-XD:N-MoRE interaction with N-core (Fig. 5, E and F). Both substitutions decreased precipitation efficiency, whereas further increasing the positive charge of the P-XD α1α2 face through an A465R enhanced the interaction, consistent with the reduced bioactivity of this mutant in the minireplicon assays. These results indicate that compensatory mutations restore virus replication in the presence of tail-truncated N by reducing binding affinity of P-XD:N-MoRE for N-core. Engineered mutations at P residues 465 and 469 showed a similar inverse correlation between biochemical interaction and polymerase activity in the context of tail-truncated N (Fig. 5G).

To assess candidate docking poses of P-XD:N-MoRE complexes to the nucleocapsid assembly, we used the data-driven HADDOCK webserver in conjunction with available structures of morbillivirus N-core [Protein Data Bank (PDB): 4UFT] (6) and P-XD-MoRE (PDB: 1T6O) (18). Because the virus evolution approach had highlighted the α1α2 face of P-XD and acidic N loop microdomains, we included orientation of residues within these domains toward each other as a mandatory spatial restraint. After two refinement steps, semiflexible minimization and molecular dynamics with explicit water layer, we obtained several top-scoring interaction poses with similar orientation. The complex represented in Fig. 5H postulates a total buried surface area of approximately 1008 Å2 and brings, as requested, residues in the acidic N loop including D154, E156, and D158 and the α1α2 P-XD face (among others, R469 and K478) in close proximity. The predicted favorable contacts are predominantly based on electrostatic interactions (−332.3 kcal/mol). Unexpectedly, this configuration postulates a second interface between solvent-exposed residues of N-MoRE associated with the docked P-XD and distinct N-core residues located in the preceding N protomer that include the flexible N-core loop spanning residues 133 to 142 (fig. S8) (6). A predicted binding affinity of 160 μM is consistent with the moderate interaction strengths detected biochemically through biolayer interferometry and affinity coprecipitation.

DISCUSSION

The C-terminal XD of paramyxovirus P protein interacts with a microdomain in the structurally largely disordered N-tail domain (13) and the L protein (14), respectively, and little was known about how these interactions could affect N-core organization. In this study, we have established a molecular link between paramyxovirus P-XD binding to MoRE located in the N-tail and subsequent interaction of P-XD with N-core, which is substantiated through physiologically relevant directed viral evolution interfaced with functional and in vitro biochemical analyses.

Quite unexpectedly, the viral evolution–based study has identified not just one, but three distinct structural domains, two located in the N protein and one in P, that can harbor point mutations restoring efficient replication of recombinant viruses lacking large sections of N-tail. Of these compensation hotspot domains, changes of residues located at the tail origin (positions 401 to 419) were the least surprising considering their immediate linear proximity to the truncation site. However, effective compensatory mutations mapped predominantly to an exposed acidic N loop on the surface of N-core (residues 146 to 161) or to a basic face of the P-XD triangular prism, formed by helices α1 and α2.

Cryo–electron microscopy models of MeV and parainfluenza virus type 5 (PIV-5) RNPs showed strong structural homologies (6, 8), indicating an overall comparable architecture. Alignment of 72 paramyxovirus N protein sequences underscored strict conservation of the acidic N loop highlighted by the CDV compensation mutations (fig. S1). Because of fuzzy appearance in MeV RNP electron density maps, this loop had been discussed as a hypothetical interaction site (5, 6), but no potential binding partner was proposed. However, anecdotal evidence implicates this section of N-core in ensuring proper polymerase function: Sendai virus (SeV) harboring an N-D153Y point mutation produced an abnormally high amount of copy-back defective interfering genomes (21), substitutions of NiV N residues 142 to 146 for alanines decreased N interaction with P (22), and we have shown that MeV N-MoRE relocation into N-core increased the relative frequency of nonproductive polycistronic viral mRNAs in infected cells (12). In each of these reports, residues targeted for substitution or N-MoRE relocation were located within, or in close proximity to, N-core residues homologous to the compensatory acidic loop identified in CDV N (fig. S9).

Transient backfolding of MeV N-MoRE onto the RNP has been proposed previously based on nuclear magnetic resonance studies of purified nucleocapsids (23). However, the nature of possible MoRE binding sites on N-core and the physiological importance of this weak interaction remained unclear. Our results demonstrate a biochemically appreciable interaction of N-MoRE:P-XD complexes with N-core, which indicates that the stable four-helix bundle configuration of MoRE complexed with P-XD is the N-core binding-competent form. Our identification of this interaction through viral evolution confirms physiological relevance for viral fitness, and five lines of evidence support the idea that P-XD forms a direct, transient interface with N-core: (i) Compensatory mutations mapped to P-XD or N-core, but not to N-MoRE; (ii) the microdomains in P-XD and N-core defined by these compensatory mutations are electronically compatible (fig. S10); (iii) engineered and natural compensatory mutations introduced into the two opposing microdomains disrupted binding in biochemical assays; (iv) purified N-MoRE in isolation was unable to efficiently interact with N; and (v) an energetically favorable docking pose could be found in silico.

Top-scoring docking poses of the P-XD interaction with the acidic loop in N-core placed P-XD–associated MoRE into contact with the flexible loop around N-core residues 133 to 142 in the preceding N monomer. We have previously physically relocated MoRE of closely related MeV into this loop, downstream of residue 138, and recovered replication-competent recombinant viruses (12). The docking poses raise the intriguing possibility that successful MoRE relocation in the previous study was achievable precisely because MoRE naturally relocates spatially into the proximity of the target site selected for transfer (Fig. 6A). Thus, the previous N engineering may have placed the relocated MoRE and, subsequently, MoRE-associated P-XD in a spatial position relative to the RNP helix trunk that mimicked the physiological arrangement.

Fig. 6 Structural conservation of interaction sites and mechanistic hypothesis.

(A) Surface representation of three protomers of morbillivirus RNP juxtaposed to RSV RNP (9) and a prediction of MeV RNP with N-MoRE relocated into N-core (12). N-core acidic loop and RSV N residues involved in P binding (24) are shown in red, and relocated MeV N-MoRE is in orange. (B) Mechanistic hypothesis of dynamic control of RdRP processivity through induced N-tail refolding and three-way interface formation between N-core and P-XD:N-MoRE. Compensatory mutations are proposed to restore proper kinetic control through reduced affinity of the interacting P-XD (green) and N-core (cyan, light marine blue) microdomains.

The interaction of C-terminal CDV P residues with N-core in the RNP assembly is reminiscent of the well-studied contacts between pneumovirus P and N. In the case of RSV specifically, the C-terminal residues of P engage in interactions with a tail-less nucleocapsid core that are modulated by electrostatic contacts (24). This N binding domain is also, in both cases, partially overlapping with the L binding domain (14, 25, 26), which brings L in close proximity to the RNA. Despite a remarkable lack of sequence conservation between paramyxovirus and pneumovirus nucleocapsids, structural alignments of RSV and CDV RNP models demonstrate that the P binding pockets in RSV RNPs are positioned in close proximity to the compensatory acidic N loop identified in the CDV RNP N-core. This strong structural mimicry implies that P-XD association with N-core identified here may reflect an ancestral step in mononegavirus negotiation of the RNP template.

To better understand the importance of this interaction for polymerase function, we wondered what the mutations identified through viral evolution may actually compensate for. Shortening of the unstructured N-tail section could, in principle, disturb the interaction through reduced positional flexibility of N-tail lacking more than 50 structurally disordered residues. In this scenario, compensatory mutations may act through increasing binding affinity. However, all substitutions emerging from virus evolution reduced electronic compatibility of the P-XD and N-core microdomains, and those tested biochemically impaired coprecipitation efficiency. These results support the alternative model that the compensatory mutations reduced affinity of the N and P domains to each other. Wild-type N-tails add considerable electron density to the outside of the RNP helix and are thought to refold upon binding of P-XD to MoRE to allow access of the polymerase complex to the template (10). This tail-ordering step modulates polymerase performance (12) and should culminate in the interaction of MoRE-bound P-XD with N-core (Fig. 6B). If the disordered N-tail residues are eliminated, reordering naturally becomes obsolete, presumably shortening the time interval between MoRE interaction with P-XD and MoRE:P-XD complex binding to N-core. We propose that substitutions reducing affinity of MoRE:P-XD for N-core compensate for this accelerated rate of MoRE:P-XD collision with N-core. In this model, N-tail refolding constitutes a regulatory element controlling polymerase dynamics. In the absence of a disordered N-tail domain, the recCDVs evolved to restore proper polymerase processivity by down-modulating affinity of the interface between P-XD and N-core, effectively replacing kinetic control of interface formation with thermodynamic regulation (Fig. 6B).

Ultimately, the formation of the three-way N-MoRE:P-XD:N-core interaction may directly contribute to allowing polymerase advance along the encapsidated template. In addition to the P-XD interaction with N-MoRE, our laboratory has recently demonstrated the interaction of a second distinct face of the P-XD trimeric prism with the polymerase L. We have shown that this interaction involves specifically one P-XD of the P tetramer, while the other three XDs are competent for N-MoRE binding (14). However, the molecular mechanism triggering iterative dissociation of the N-MoRE:P-XD and L:P-XD interaction, which emerged as a prerequisite for polymerase mobility, remained elusive. Here, we show the interaction of the third P-XD prism face with N-core, placing P-XD at a pivotal position in between the RNP, N-tails, and the polymerase protein. Residue R465 in MeV P-XD has been shown to act as a determinant for the stability of the P-XD triple helix assembly through the formation of salt bridges between helices α1 and α2 (27). On the basis of the placement of P-XD at the center of multiple transiently forming interfaces and this demonstrated effect of electronic interactions on P-XD conformational stability, we propose that the assembly of the electrostatic complex with N-core identified in this study triggers a switch from the stable triple helix P-XD configuration to a less-ordered intermediate. This structural reorganization is anticipated to alter binding affinities to N-MoRE and/or L, enabling temporary separation of P-XD from its binding partners as proposed (14) and advance of the polymerase complex along the template by one N protomer. Spontaneous restoring of the stable triple helix conformation once P-XD is removed from the destabilizing electronic influence of the N-core binding site sets the stage for rebinding to L (14) and N-MoRE of a downstream N protomer followed by repeat of the cycle. In this mechanistic model, the N-core to P-XD interface thus functions as a molecular checkpoint that governs polymerase mobility through structural modulation of the P-XD triple helix configuration.

MATERIALS AND METHODS

Cell culture

African green monkey kidney epithelial cells [CCK-81; American Type Culture Collection (ATCC)] stably expressing canine signaling lymphocytic activation molecule (Vero-cSLAM) (28) and baby hamster kidney cells (C-13; American Type Culture Collection) stably expressing T7 polymerase (BSR-T7/5) (29) were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 7.5% fetal bovine serum and were reselected with geneticin (100 μg/ml) at every fifth passage. Transient transfections were performed with GeneJuice (Novagen) according to the manufacturer's protocol.

Molecular biology

Plasmids encoding expression constructs of CDV strain Onderstepoort N, P, and L (16), morbillivirus minigenome (10), and plasmids harboring full-length cDNA copies of the CDV strain 5804PeH genome (16) were previously described. Construction of the nucleoprotein truncation [425–479] without the extra residues resulting from the original cloning strategy was removed by site-directed mutagenesis. For protein production, the sequence of CDV N strain 5804 encompassing residues 1 to 419 was amplified by polymerase chain reaction (PCR) with a C-terminal hexahistidine tag and an enterokinase cleavage site (QDGNDDDDKLEHHHHHH) and cloned onto plasmid pET30a+. To express GST-XD, the residues of P strain 5804 [457–507] were amplified by PCR and cloned in pGEX 6P1 downstream of the GST and thrombin cleavage sequence between Bam HI and Eco RI. Extra C-terminal residues from the cloning strategy resulted in the added C-terminal residues GSGSQL. To express the fusion GST-XD-box2 containing N-MoRE, residues 486 to 505 of N strain 5804 were directly fused downstream the GST-XD sequence, with residues GS and QL. All substitutions were engineered by site-directed mutagenesis, and sequences were verified by Sanger sequencing (Genewiz).

Minigenome luciferase reporter assay

BSR-T7/5 cells (5000) were seeded in a clear bottom white wall 96-well plate and transfected upon reaching 60% confluence with plasmids encoding the morbillivirus luciferase replicon reporter (0.044 μg), CDV-N (0.016 μg), and a twofold serial dilution of CDV-L and CDV-P from 0.02 μg each. The total amount of DNA transfected was adjusted with a pCDNA3.1 plasmid (Invitrogen). Specifically, for each experiment, plasmids were combined in master mixes containing all components, but the plasmid encoding the mutation-bearing protein was investigated. These master mixes were then serially diluted five times in twofold steps with a mix of pCDNA3.1 and replicon reporter, keeping the total DNA amount and the amount of reporter plasmid equal in every dilution step. Each dilution was finally mixed with the plasmid encoding the mutation-bearing protein investigated. In the case of P mutations, master mixes were further prepared with either CDV-N or CDV-NΔ[425–479]. For each experiment, plates were seeded in parallel, and transfections were carried out with a master mix of GeneJuice transfection reagent (MilliporeSigma) according to the manufacturer's instructions. Each experiment included a reference plate containing a serial dilution of the unmutated plasmid for normalization. Firefly luciferase activities were determined 24 hours after transfection in a Synergy H1 microplate reader (BioTek) using ONE-Glo luciferase substrate (Promega). Relative RdRP activities (relA) were determined according to % relA = (experimental)/(peak signal) × 100, with peak signal corresponding to the measured peak activity of the reference dilution of the unmutated plasmid. All experiments were performed in at least three independent replicates, each measured in three dependent repeats.

Virus recovery

Recombinant CDVs were recovered in six-well plates previously seeded with 2 × 105 BSR-T7/5 cells per well by transfecting at 60% confluence 1.25 μg of the cDNA copy of the modified genome and MeV helper plasmids (10) IC-B-N (0.1 μg), IC-B-P (0.3 μg), and IC-B-L (0.3 μg). All recombinant CDV genomes harbored an additional transcription unit encoding the mKate fluorescent protein positioned between H and L ORFs (16). Each well of transfected cells was overlaid 48 hours after transfection onto a whole six-well plate of Vero-cSLAM previously seeded with 2 × 105 cells per well, and cells were monitored for the emergence of syncytia. Every other day, infected cells from one well were either harvested if CPE had reached approximately 80% or passaged after dilution into fresh six-well plates. For reinfections, cell-associated viral particles were released through two freeze/thaw cycles, and cleared supernatants (5 min, 1000g, 4°C) were transferred in serial dilutions to fresh Vero-cSLAM cells. Infected cells that showed >80% CPE in less than 48 hours were not passaged further to prevent the buildup of defective interfering particles. Recombinant virus RNAs were extracted with the ZR Viral RNA Kit (Zymo Research), and cDNA copies were generated using random hexamer primers and SuperScript III reverse transcriptase (Invitrogen). Modified genome regions were amplified using appropriate primers and subjected to Sanger sequencing (Genewiz).

Characterization of recombinant virus early replication profile

Three independent transfections for each rescue event were overlaid on a Vero-cSLAM–seeded six-well plate each. Three syncytia were randomly picked up and added onto a well of fresh cells. Emerging syncytia were counted at 48 hours post-infection. Syncytium size was determined by estimating the total number of cells in microphotographs (×100 magnification) based on the average count of cells per 100 square pixels (n = 10) in GIMP 2.0. The surface area covered by a syncytium on each picture was determined with GIMP 2.0 and expressed as the estimated number of fused cells.

Preparation of virus stocks

CDV virus stocks were prepared by infecting Vero-cSLAM cells at a multiplicity of infection of 0.01, followed by incubation at 37°C for 40 to 48 hours. When microscopically observed virus-induced CPE reached approximately 90%, cell-associated viral particles were released through two consecutive freeze/thaw cycles, followed by a clearance spin (1000g, 5 min), and storage of single-use aliquots at −80°C. Viral titers were determined by TCID50 (median tissue culture infectious dose) titration on Vero-cSLAM cells as described (30).

Multistep virus growth curves

A 12-well plate of Vero-cSLAM cells was infected at a multiplicity of infection of 0.001, followed by incubation at 37°C for 60 hours. At 12, 18, 24, 36, 48, and 60 hours post-infection, one well of infected cells was harvested and titrated by end-point dilution assay. Each time point consisted of three independently infected cell monolayer.

Sequence alignments

Alignments were conducted using T-Coffee PSI-Coffee server and formatted with ESPript (31).

Protein homology modeling

Modeling of CDV nucleoprotein (5804P strain) assembly was conducted using SWISS-MODEL server on expasy.org and MeV nucleoprotein assembly structure (PDB: 4UFT) (6). Modeling of SeV Cantell strain clone cCdi (AB855654.1) and CDV (5804P strain) RNA-free nucleoprotein monomers was conducted using SWISS-MODEL server on expasy.org using MeV RNA-free nucleoprotein structure (PDB: 5E4V) (5). Modeling of CDV, HPIV-3, NiV, and PIV-5 phosphoprotein XDs was conducted using SWISS-MODEL server on expasy.org using MeV (PDB: 1OKS) (17), SeV (PDB: 1R4G) (32), HeV (PDB: 4HEO) (33), and MuV (PDB: 3BBZ) (34) P-XD crystal or solution structure. Modeling of MeV nucleoprotein with internal N-MoRE (12) was performed on I-TASSER server (35) using additional restraints with α-helix secondary structure specified for residues of MoRE (QDPQDSRRSADALLRLQAMAGI) and using the docked pose of P-XD-MoRE (PDB: 1T6O) on MeV nucleocapsid (PDB: 4UFT) as additional template (see the molecular docking method).

Molecular docking

N-core (PDB: 4UFT) (6) and P-XD-MoRE (PDB: 1T6O) (18) complexes were generated using HADDOCK webserver (36). HADDOCK uses ambiguous interaction restraints to guide the first steps in the docking process. Active and passive residues were defined on the basis of recCDV N[425–479] compensatory mutations and surrounding residues, respectively, using their equivalent residues after sequence alignment with MeV sequences (N active residues: D154, E156, and Q158; N passive residues: E148 and F209; P active residues: R465, K469, K478, and M482; P passive residues: R472 and D486). Initially, 1000 docking poses were generated with rigid body docking, and the best 200 poses were then refined by a semiflexible simulated annealing in torsion angle space stage. This is a three-step molecular dynamics–based refinement. This stage was followed by an explicit water refinement stage with short (2-fs steps) molecular dynamics simulation at 300 K. The final scoring combined electrostatics, desolvation, and van der Waals energy terms in combination with the energetic contribution of the restraint used to drive the docking. Binding affinity prediction was performed using the PRODIGY server (37).

Recombinant protein expression and purification

GST fusion proteins were expressed in competent One Shot BL21 pLysS chemically competent Escherichia coli (Thermo Fisher Scientific). Cells were grown at 37°C for 8 hours or until saturation, and protein expression was induced for 16 hours at 28°C by adding one volume of LB broth with ampicillin (100 μg/ml) and 0.33 mM final isopropyl-β-d-thiogalactopyranoside (IPTG). Cells were pelleted and lysed in GST lysis buffer containing 50 mM tris-HCl (pH 7.4) (room temperature), 150 mM NaCl, 0.2% Triton X-100, a cocktail of protease inhibitor (Pierce, Thermo Fisher Scientific), and chicken egg white lysozyme (1 mg/ml) for 1 hour at room temperature. Lysates were treated with Pierce universal nuclease (Thermo Fisher Scientific), clarified by 30-min centrifugation at 15,000g and 4°C, and incubated overnight at 4°C with Glutathione Sepharose 4B beads (GE Healthcare). Beads were washed three times with 20 volumes of wash buffer containing 50 mM tris-HCl (pH 7.4) (room temperature) and 150 mM NaCl and stored at 4°C.

For recombinant nucleoprotein purification, BL21 E. coli transformed with pET N[1–419] or pET N[1–419]-E156K were grown at 37°C for 8 hours or until saturation and protein expression was induced for 16 hours at 20°C by adding one volume of LB broth with kanamycin (50 μg/ml) and 0.33 mM (final concentration) IPTG. Cells were pelleted, frozen, and lysed in N lysis buffer containing 50 mM tris-HCl (pH 7.4) (room temperature), 450 mM NaCl, 1 mM MgSO4, a cocktail of protease inhibitor (Pierce, Thermo Fisher Scientific), and chicken egg white lysozyme (1 mg/ml) for 1 hour at room temperature. Lysates were treated with Pierce universal nuclease (Thermo Fisher Scientific) and clarified by centrifuging twice at 15,000g and 4°C for 30 min. Clear lysates were mixed with Ni-NTA Superflow resin (Qiagen) for 1 hour with 20 mM imidazole and washed three times with 20-bed volumes of 50 mM tris-HCl (pH 7.4) (room temperature) and 450 mM NaCl with 60 mM imidazole. Proteins were eluted two times with one volume of wash buffer containing 500 mM imidazole, followed by buffer exchange for phosphate-buffered saline (PBS) using PD-10 desalting columns with Sephadex G-25 resin (GE Healthcare).

Affinity coprecipitations

To allow protein interactions, ~200 ng of purified N[1–419] or N[1–419] E156K was mixed for 1 hour at room temperature and 1000 rpm shaking, with ~100 μg of purified GST, GST-MoRE, GST-XD, GST-XD R469E, or GST-XD–MoRE (with or without substitutions A465V, A465R, R469G, or R469E) immobilized on 20 μl of Glutathione Sepharose 4B beads (GE Healthcare). Beads were washed three times with 10-bed volumes of PBS. Proteins were eluted with two-bed volume of 1× Laemmli buffer, and 10 μl was analyzed through SDS–polyacrylamide gel electrophoresis on 12% gels, followed by staining with R250 Coomassie blue. Densitometric analysis was performed using scanned gels with Fiji software (38). For Western blot analysis, gels were blotted on polyvinylidene difluoride membranes (Millipore) with semidry transfer and subjected to chemiluminescence detection using monoclonal antibodies directed against the CDV nucleoprotein (clone DV2-12, Invitrogen). Immunoblots were developed using a ChemiDoc digital imaging system (Bio-Rad) for image visualization.

Biolayer interferometry

GST (± XD or XD-MoRE, ± R469E)–coupled Biosensors (ForteBio) were dipped into increasing concentrations (0.5 to 4 μM) of recombinant nucleoproteins for 600 s in PBS–0.01% bovine serum albumin–0.002% Tween 20 and then dipped back into kinetic buffer in the absence of recombinant nucleoproteins for 600 s to follow dissociation. Reference sensors with GST were used to control for nonspecific binding. Real-time binding kinetics were analyzed and calculated using the Octet Red software package.

Statistical analyses

Experimental variation and statistical significance of differences between sample means were assayed using either one- or two-way analysis of variance (ANOVA) or nonparametric Kruskal-Wallis test, with appropriate post hoc tests as specified in the figure legends, using the Prism 8 (GraphPad) software package. In all experiments, the significance level α was set to <0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. For initial setup of the minireplicon ratio test and virus spread quantitation, datasets were pretested for normal or lognormal distribution using Anderson-Darling, D'Agostino-Pearson, and Shapiro-Wilk and Kolmogorov-Smirnov tests. For clarity, data variation from independent repeats was represented either directly when possible by a symbol for each data point or with error bars representing geometric SD to the mean.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/10/eaaz1590/DC1

Fig. S1. Multisequence alignments of N and P proteins of representative members of the paramyxovirus family.

Fig. S2. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

Fig. S3. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

Fig. S4. Genetic stability of recombinant viruses harboring rebuilt compensatory mutations.

Fig. S5. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

Fig. S6. Minigenome activity profiles of full-length (blue) and tail-truncated (red) N.

Fig. S7. Expression and purification of recombinant N and P protein subsets.

Fig. S8. Molecular docking of P-XD:N-MoRE complexes into N-core.

Fig. S9. Position of the acidic N-core loop in distinct paramyxovirus N proteins and activity associations.

Fig. S10. Spatial organization and electronic properties of P-XD in crystal structures solved for MeV, SeV, HeV, and MuV and corresponding homology models generated for CDV, HPIV-3, NiV, and PIV-5.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

Acknowledgments: We thank M.-S. Russ for technical assistance, V. von Messling for the CDV reverse genetics system, Y. Yanagi for Vero-cSLAM cells, and K. K. Conzelmann for BSR-T7/5 cells. Funding: This work was supported, in part, by Public Health Service grants AI071002 and AI141222 from the NIH/NIAID (to R.K.P.). Author contributions: J.S.: conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing (original draft preparation), writing (review and editing), and responsible for Figs. 1 to 5 and all the Supplementary Materials. V.D.T.: investigation; R.M.C.: investigation and writing (review and editing); R.K.P.: conceptualization, formal analysis, funding acquisition, validation, project administration, supervision, writing (original draft preparation), writing (review and editing), and responsible for Figs. 1 to 5 and all the Supplementary Materials. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data are available from the authors upon request. The CDV reverse genetics and minigenome systems and expression plasmids of CDV polymerase components described in this study can be provided by the corresponding author pending scientific review and a completed material transfer agreement from Georgia State University. Requests for these materials should be submitted to the corresponding author.

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G7 pledges to mediate coronavirus fallout as US fed slashes rates - Aljazeera.com

Posted: 04 Mar 2020 08:50 PM PST

G7 finance officials pledged on Tuesday to act to safeguard against risks posed by fast-spreading coronavirus as the United States Federal Reserve implemented its first -and biggest- surprise interest rate cut since the 2008 financial crisis.

The move came as G7 finance ministers held a phone call to discuss how to hold up the economy and markets, the latter of which last week saw its worst week since 2008. Speaking to reporters after the call, Japanese Finance Minister Taro Aso said that "anything will be included, both monetary and fiscal steps" to support price stability. 

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"We reaffirmed our commitment to adopt all appropriate policy steps to protect the economy from downside risks posed by the coronavirus, and that we stand ready to cooperate further on timely and effective measures," Aso said.

German Finance Minister Olaf Scholz also said the G7 had "all means" at its disposal. "Should the need arise, we have all the means to counter a global downturn," Scholz said in a statement on Twitter.

Global stocks suffered a rout last week on fears that the disruption to supply chains, factory output and global travel caused by the epidemic could deal a serious blow to a world economy trying to recover from the US-China trade war.

The coronavirus, which emerged in the central Chinese city of Wuhan late last year, has spread around the world over the past week, with more new cases now appearing outside China than within. There are more than 90,000 cases globally, with more than 80,000 of them in China, and infections appearing in 77 other countries and territories, with Ukraine the latest country to report its first case.

After what critics said was an initially hesitant response, China imposed sweeping restrictions, including suspensions of transport, sealing off communities, and extending a Lunar New Year holiday across the country.

Now, China is increasingly concerned about the virus being brought back into the country by citizens returning from new hot spots elsewhere. Authorities on Tuesday asked overseas Chinese to reconsider or minimise their plans to travel home. All travellers entering Beijing from South Korea, Japan, Iran and Italy would have to be quarantined for 14 days, a city official said. Shanghai has introduced a similar order.

The most serious outbreak outside China is in South Korea where President Moon Jae-in declared war on the virus, ordering additional hospital beds and more masks as cases rose by 600 to nearly 5,000. Thirty-four people have died in South Korea.

In the United States, where the Federal Reserve announced it was slashing interest rates by a half a percentage point on Tuesday, the virus is now believed to be present in at least four communities in the Pacific Northwest. Six people have died in the Seattle outbreak. The US Centers for Disease Control and Prevention lists more than 90 cases across the US.

Iran reported infections rising to 1,501, with 66 deaths, including a senior official. The death toll in Italy jumped to 52 on Monday from 34 the day before and the total number of confirmed cases in Europe's worst-affected country climbed past the 2,000 mark. Germany reported 31 new infections, taking its tally to 188.

SOURCE: Reuters news agency

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Latinos and Young People Come Through for Bernie Sanders in California Primary - The New York Times

Posted: 04 Mar 2020 08:50 PM PST

LOS ANGELES — California appeared to deliver a pivotal victory to Senator Bernie Sanders Tuesday night, driven by a coalition of Latinos, young people and liberal voters up and down the state. With 415 pledged delegates, California is by far the biggest prize of Super Tuesday and Mr. Sanders had been banking on a significant win there.

With millions of ballots left to count, it is impossible to know how the delegates will be allocated, and it may not be clear for several days or even weeks. Roughly two-thirds of California delegates are distributed based on congressional districts, with candidates needing at least 15 percent of the vote to win any delegates.

But with Mr. Sanders receiving more than 70 percent of Latino voters under the age of 30, and about half of Latino voters over all, according to exit polls, his lead over the other candidates looked decisive.

The Associated Press projected Mr. Sanders as the winner of the state just minutes after the polls closed, while thousands of voters in Los Angeles County were still in line to cast their ballots because of problems with voting machines.

A new $300 million voting system caused waits as long as four hours at dozens of polling sites throughout Los Angeles County, including in Westwood, the San Fernando Valley, Los Feliz and the east side of Los Angeles. The sign-in process to check voters against the voting rolls took hours at several sites, and internet issues left many centers with a small fraction of working machines. Lines continued past 10 p.m. at many locations, including several college campuses.

More than 4 million mail-in ballots had been sent in by Tuesday morning, and as many as 5 million ballots were left to count by Wednesday, according to an analysis from Political Data Inc., a California-research group that closely tracks returns.

Along with Mr. Sanders, both former Vice President Joseph R. Biden Jr. and Michael R. Bloomberg, the former New York mayor, seemed likely to receive delegates in the state, based on early returns. Mr. Bloomberg had poured $66 million into television advertising here, far more than any other candidate.

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State officials moved up the state's primary to March this year in an attempt to make California more politically influential when it comes to choosing the Democratic presidential nominee. The change meant that voters saw far more television advertisements: roughly $120 million worth, with the majority coming from Mr. Bloomberg.

Several candidates visited parts of the state that have long been ignored in statewide elections, including Bakersfield and Riverside. Still, California never drew the candidates of early-voting states like Iowa or New Hampshire and most of the candidates relied on national news coverage to gain recognition in the state.

There was a clear generational split in the state, with Mr. Sanders winning among voters under 49, but Mr. Biden was the clear preference with voters older than 50. Mr. Biden won among older voters, black voters and moderates in California, the same coalition that helped him win several other states Tuesday. Mr. Sanders appeared to win among voters at all education levels, according to exit polls.

Mr. Sanders won more white and Asian-American voters, but reflecting a national trend, Mr. Biden took the lead among black voters, according to exit polls. Voters who chose a candidate in the final days sided with Mr. Biden by a 10-point margin, those polls showed.

In 2016, California proved to be something of a firewall for Hillary Clinton, who beat Mr. Sanders with 53 percent of the vote to his 46 percent. That year, the state voted in June, when the primary race was all but over.

This year it was Mr. Sanders who counted on California to be his own firewall. His campaign frequently referred to the "first five" states, lumping California with Iowa, New Hampshire, Nevada and South Carolina. In turn, the campaign poured significant resources into advertising and organizing in a state that has traditionally been viewed as impossible to penetrate by door knocking because of its vast size.

Mr. Sanders also held several large rallies in California, including one at Venice Beach in December with Representative Alexandria Ocasio-Cortez of New York, and several in the final days before Tuesday's vote.

"I'm cautiously optimistic that later in the evening, we can win the largest state in this country, the state of California," Mr. Sanders said during a rally in Vermont, before the polls had closed in California.

For months, the Sanders campaign focused its efforts in heavily Latino neighborhoods around the state, from the Coachella Valley and Bakersfield to Santa Ana, as well as in South and East Los Angeles.

For Lorena Vellanowth, Mr. Sanders clearly showed a commitment to issues that she believes are important to Latino voters. Ms. Vellanowth came to Los Angeles from Mexico as a baby, and her daughter went on to graduate from U.C.L.A., and then to attend graduate school at the University of Southern California.

But all that success came with a big caveat: Her daughter, who works in public administration for the city of Anaheim, is struggling under the weight of almost a half-million dollars in student loans.

"Bigger than my mortgage," said Ms. Vellanowth, 43, who works in health care. She said her daughter is getting married this month, and she worries about how her daughter and husband will ever be able to afford having children, or buy a home.

That reason alone was enough to draw her, and many of her family members and friends in the Latino community in Los Angeles to Mr. Sanders, attracted, she said, by his message of fighting the type of inequality they feel every day.

"I'm Latina, and you'd think that's not someone I'd vote for, an old white guy," she said on Monday afternoon, standing outside a polling place at a park in the heavily Latino East Los Angeles named for Ruben Salazar, a Los Angeles journalist who was killed in 1970 during a protest against the Vietnam War.

But she worries about her candidate's future, now that the establishment of the Democratic Party is coalescing around Mr. Biden.

"I just hope the Democratic Party doesn't screw him," she said, of Mr. Sanders. Ms. Vellanowth, who became an American citizen in 2013 and cast her first presidential vote for Mr. Sanders in the 2016 primary, said she feared history would repeat itself, with the moderates of the party gathering around Mr. Biden as they did with Hillary Clinton in 2016, thwarting Mr. Sanders's candidacy.

Mr. Sanders also captured the imagination of California's young people, many of whom are struggling with the exorbitant cost of living in the state and buckling under student loans.

"It's called a living wage for a reason," said Alex Mora, 24, who lives in East Los Angeles, and voted for Mr. Sanders because of his promise to raise the minimum wage. "A lot of us are stuck at home and we can't find a way out because of how low paying our jobs are."

In California, which has the fifth-largest economy in the world but is also home to the highest poverty rate in the nation when housing costs are factored, wealth inequality has been a paramount political issue driving voters to the polls.

With such a wealth chasm as a backdrop, Mr. Sanders's platform of "Medicare for all," free college tuition and student loan relief has resonated deeply. He has also promised to protect so-called dreamers — immigrants who arrived in the country illegally as children — and roll back the Trump administration's executive orders on immigration.

It's not only California's poor and working class who have supported Mr. Sanders; some who may not struggle financially have been moved by the stark inequality they see every day.

"He's the one who seems like he's most likely to improve the quality of life for the average person," said Michael Lussier, 29, who works in real estate.

Mr. Lussier lives in downtown Los Angeles, which has been engulfed by a worsening homeless crisis. "Living so close to all that makes it harder to vote for anyone who will perpetuate the status quo," he said.

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California announces first coronavirus death, bringing US fatalities to at least 11 - CNBC

Posted: 04 Mar 2020 08:50 PM PST

Shelves where disinfectant wipes are usually displayed is nearly empty at a Target store on March 02, 2020 in Novato, California.

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Local health officials in California announced Wednesday the state's first COVID-19 death, bringing U.S. fatalities to at least 11.

The patient was an elderly adult with underlying health conditions, county officials said, adding that the person was the second confirmed COVID-19 case in the county and is now the first death related to the virus in the state.

"We extend our deepest condolences to the loved ones of this patient," Placer County Health Officer Dr. Aimee Sisson said in a statement. "While we have expected more cases, this death is an unfortunate milestone in our efforts to fight this disease, and one that we never wanted to see."

County officials said the patient was likely exposed during international travel from Feb. 11 to Feb. 21 on a Princess cruise ship that departed from San Francisco and traveled to Mexico. Placer County officials said the patient arrived at Kaiser Permanente Roseville Medical Center by ambulance on Feb. 27 and was in isolation at the hospital.

As of Wednesday, there are 53 confirmed cases of COVID-19 in California, including 24 from repatriation flights. According to the Centers for Disease Control and Prevention, California has more cases of the virus than any other state.

Placer County, which is in northern California near Sacramento, is monitoring ten health care workers from the hospital, the county said, and five emergency responders who were exposed prior to the patient being placed in isolation. The county said none of them are exhibiting symptoms. The county said other passengers from the cruise may have also been exposed.

"While most cases of COVID-19 exhibit mild or moderate symptoms," Dr. Sisson added, "this tragic death underscores the urgent need for us to take extra steps to protect residents who are particularly vulnerable to developing more serious illness, including elderly persons and those with underlying health conditions."

The news comes after Vice President Mike Pence, who is leading the U.S. response to the COVID-19 outbreak, announced the country's tenth death. CNBC has confirmed that the tenth death was in Washington state, where all of the first nine deaths occurred as well.

"The state is working with federal officials to follow up on contact tracing of individuals that may have been exposed to provide treatment and protect public health," California Gov. Gavin Newsom said. "This case demonstrates the need for continued local, state and federal partnership to identify and slow the spread of this virus."

Earlier Wednesday, Los Angeles-area officials announced six new COVID-19 cases in the county over the past 48 hours, prompting that southern California county to declare a local emergency to help free up federal and state funding. Officials said at the announcement that there were no community spread cases in Los Angeles county, meaning that all cases could be traced back to a known or suspected source. 

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Mobile port widening project moving along - NBC 15 WPMI

Posted: 04 Mar 2020 08:46 PM PST

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Mobile port widening project moving along  NBC 15 WPMI

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Florida lawmakers pass measure after viral video of officers arresting 6-year-old - Fox News

Posted: 04 Mar 2020 08:18 PM PST

After a gripping video of a 6-year-old being hauled out of her school in handcuffs circulated last month, Florida lawmakers passed a bill Wednesday to require law enforcement to put in place procedures for arresting children under 10.

The amendment, dubbed the "Kaia Rolle Act" after the crying young girl in the video, was added into a school safety bill spawned by the Parkland high school shooting and adopted unanimously.

Last month, Kaia Rolle's family made public footage from a police officer's body camera, sparking outrage from the public and lawmakers. "Please help me, please just let me go," the girl can be heard pleading with the arresting officer. "I don't want to go in the police car, no please, please," Rolle begged. She can be heard asking officers for a "second chance," after the arrest for a temper tantrum she threw at school.

Kaia watched from the gallery of the Florida House with her grandmother Meralyn Kirkland as lawmakers stood with her in solidarity to approve the legislation.

"Asking for a second chance is a universal principle that is found at the core of our existence here in this country," said Rep. Kionne McGhee, the Democratic leader in the House who sponsored the measure in his chamber.

McGhee told lawmakers there were too many young children being arrested at schools and it was "incumbent upon us at this very moment, at this very hour, to answer the call that young Kaia rendered when she was being arrested."

CALIFORNIA MAN GETS 5 YEARS IN PRISON FOR HARASSING PARKLAND SCHOOL SHOOTING VICTIMS 

The sponsor of the original bill, Republican Ralph Massullo, called McGhee's change to the bill a "friendly" one, sparking applause throughout the chamber.

Kirkland expressed gratitude for the overwhelming support from lawmakers.

"I needed people to know that there is this law in the books that allows our babies to be arrested. It was not a one-off with Kaia. It can happen to any child and any family out there, and that we needed to make a change," the grandmother said.

The bill will now have to pass on the Senate floor.

FLORIDA PAIR ARRESTED AFTER 118 DOGS RESCUED FROM 'WORST CASE OF ANIMAL HOARDING, NEGLECT' EVER SEEN 

"We just can't just sit here and sympathize and empathize, or send love or send our regrets," Kirkland said. "We've got to do something to stop this."

In September, officers arrested the first-grader at her charter school, Lucious & Emma Nixon Academy, after school officials said she kicked and punched staff members. She was charged with misdemeanor battery and ordered to appear before a judge, but the charges were later dropped.

"She's going to have to come with us now," Officer Dennis Turner tells a school employee, video shows. "Stand up, stand up … come over here."

"What are those for?" Kaia asks of the zip ties the officer used to bind her wrists. "It's for you," says Turner. The officer was later fired for not receiving supervisor approval to arrest a child under 10.

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After Kaia was taken away for fingerprints and a mugshot, Turner reentered the school to tell officials this was not the first child he had arrested. "How old was she, 8?" he asked. School officials informed him she was 6. "That would make her the youngest," Turner said.

The Associated Press contributed to this report. 

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With Mike Bloomberg out of presidential race, Bloomberg journalists resume normal political coverage - CNBC

Posted: 04 Mar 2020 07:50 PM PST

Democratic presidential candidate Mike Bloomberg holds a campaign rally on February 4, 2020 in Detroit, Michigan.

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With Mike Bloomberg out of the presidential race, Bloomberg LP reporters are free to investigate the Democrats still vying for the spot, Editor-in-Chief John Micklethwait told employees in an email Wednesday afternoon.

"Now that Mike has said he is leaving the race for President, we will return to our normal coverage of the election; we will follow exactly the same coverage rules for the Democratic presidential candidates and President Donald Trump," Micklethwait wrote to Bloomberg's editorial and research staff in a memo that was obtained by CNBC.

Mike Bloomberg is the founder and 89% shareholder in Bloomberg LP, a financial software company that owns Bloomberg News. When Bloomberg announced he was joining the race in November, Micklethwait said the company would continue its "tradition of not investigating Mike (and his family and foundation )" and would "extend the same policy to his rivals in the Democratic primaries."

The decision was criticized by some, including President Donald Trump.

The former New York mayor earlier Wednesday dropped out of the presidential race after a poor performance in the Super Tuesday contests and immediately endorsed former Vice President Joe Biden's candidacy.

Micklethwait said in the memo the company will "disclose Mike's financial support for other Democrats — just as we have always done where his financial support for political causes is relevant to our reporting."

He also highlighted the reporting the company's journalists have done since Bloomberg's decision to join the race.

"As I pointed out back on November 24th, we found ourselves in an unprecedented situation: no other newsroom of our size has had to deal with anything similar. Since then, we have written around 1,100 articles on the contest — and that does not include all the broadcast pieces and interviews, nor all the third party articles we have made available to our terminal customers."

Read the full memo:

Now that Mike has said he is leaving the race for President, we will return to our normal coverage of the election; we will follow exactly the same coverage rules for the Democratic presidential candidates and President Donald Trump. We will disclose Mike's financial support for other Democrats — just as we have always done where his financial support for political causes is relevant to our reporting.

I would like to pay tribute to everybody who has covered the campaigns — and the independent way that we have reported the race. As I pointed out back on November 24th, we found ourselves in an unprecedented situation: no other newsroom of our size has had to deal with anything similar. Since then, we have written around 1,100 articles on the contest — and that does not include all the broadcast pieces and interviews, nor all the third party articles we have made available to our terminal customers. You can see an audit of our coverage up to March 2 on our website and the terminal, which we will continue to update. But I would like to thank everybody for all the hard work that this has involved — and look forward to what lies ahead.

 John

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Wall Street, Encouraged by Biden’s Wins, Breaks Out Its Checkbooks - The New York Times

Posted: 04 Mar 2020 07:20 PM PST

After weeks of worrying that Senator Bernie Sanders would trounce his rivals on Super Tuesday and set the tone for a rancorous Democratic nomination, Michael Novogratz, the longtime trader and merchant banker, was newly motivated on Wednesday morning.

Joseph R. Biden Jr., the former vice president and a moderate candidate with solid support on Wall Street, had notched an unexpectedly strong showing in the Super Tuesday primaries. Of course, Mr. Sanders could still win the nomination — but for Mr. Novogratz and others on Wall Street, Mr. Biden's resurgence offered the opportunity to bankroll a Democrat they can get behind.

"There's a lot of momentum on Biden's side," Mr. Novogratz said. Already, one of his colleagues at Galaxy Digital, a cryptocurrency investment firm, was planning a Biden fund-raiser. Mr. Novogratz himself was set to host a dinner Wednesday night to raise money for voter-registration efforts in swing states. Stocks, hammered in recent days over fears of the coronavirus — along with an undercurrent of worry about Mr. Sanders, according to some traders — were rallying as he spoke on Wednesday morning. Between the easing of interest rates and the improving political picture, Mr. Novogratz said, "we're getting some bounce."

By the end of the day, the S&P 500 had risen more than 4 percent, bouncing back from a steep drop the day before, reflecting Wall Street's relief about Mr. Biden's success.

For moderates on Wall Street, this election season has been stressful. Accustomed to being courted by candidates seeking their financial support, bankers, investors, traders and executives from hedge funds and private equity firms have struggled to feel wanted in 2020. Mr. Sanders is an avowed democratic socialist who wants to tax billionaires — of which there are plenty on Wall Street — and has eschewed big donations. Michael R. Bloomberg, the former New York mayor and a multibillionaire respected by many financiers, didn't want their money either. Pete Buttigieg, the former South Bend, Ind., mayor and onetime McKinsey consultant whose common-sense centrism had intrigued others, had lost ground in recent weeks. And until this past weekend, Mr. Biden's path to the Democratic nomination appeared tenuous.

Against that backdrop, Tuesday night's results brought a surge of relief to many on Wall Street, who — fearful of the anticapitalist sentiment espoused by Senator Sanders and wary of another four years of a polarizing President Trump — had essentially sidelined themselves from political activism. Now, with Mr. Biden's victories presenting him as an effective political foil for Mr. Sanders, executives who had shrugged off the race are racing to support a candidate whom they believe can defend the social policies they are committed to without upending the economy.

Tracy V. Maitland, the president of Advent Capital Management and a supporter of Mr. Biden's, said he was fielding numerous calls and texts from elated friends on Wall Street and in the entertainment industry. "People think we've turned a corner," he said.

Even Leon Cooperman, the billionaire who had been supporting Mr. Bloomberg until he dropped out, expressed some relief. "I'm happy to see the country is not inclined towards electing a communist or a socialist," Mr. Cooperman said.

Their objections to Mr. Sanders, whose signature policy was a universal, government-sponsored health care plan called Medicare for All, were reactions to both his style and his substance. His harsh talk — that aspects of the deceased Cuban dictator Fidel Castro's regime were admirable, and that billionaires shouldn't exist — unsettled many. And his embrace of expensive, big-government social programs like health care and college-debt forgiveness, combined with new wealth taxes and a desire to more aggressively regulate lending and banking, felt to many Wall Streeters like an assault on their way of life.

"I don't like assassination by categorization," Lloyd C. Blankfein, the former chief executive of Goldman Sachs, said of Mr. Sanders in a recent interview with The Financial Times. Other finance executives, who felt similarly but were reluctant to go public with their own views for fear of alienating clients or employees, said they were relieved Mr. Blankfein spoke up.

Many Wall Street executives don't think Mr. Sanders's policies are sound. "Canceling all college debt will help relatively rich and high-earning university graduates, versus the money coming out of the pocket of everyone else, including those who never went to university and therefore earn less," Jennifer Fan, a former hedge fund manager who now invests her own money, said of Mr. Sanders's policies. "The ideas are just not good, the consequences aren't well thought out."

By contrast, Mr. Trump's economic policies, which have rolled back regulations, slashed taxes and focused on job creation, have favored corporations and coincided with a booming economy. Yet many Wall Street moderates are loath to support him, too. The president's tough immigration stance has rattled bankers and traders, many of whom travel widely and depend greatly on foreign-born employees to bolster their ranks. His apathy toward climate change, whose effects are increasingly driving investing and spending decisions, has concerned many. And even in an industry once known for its boorish behavior and treatment of women, Mr. Trump's meanspirited rants toward the Federal Reserve chair, the late Senator John McCain, and scores of other prominent public figures have landed badly.

Still, Wall Street's embrace of Mr. Biden could have downsides for the candidate. Hillary Clinton took in millions of dollars in campaign contributions from bankers and hedge fund managers. But during the 2016 election cycle, her private speeches to Goldman Sachs, including a remark that blaming the global banking system for the financial crisis was an "oversimplification," drew scathing attacks from both Mr. Sanders and Mr. Trump, who argued separately that she was too beholden to Wall Street. Mitt Romney, who spent decades as a private-equity executive at Bain Capital before running for president in 2012, was attacked for the debt and layoffs his firm imposed on its investment companies during his tenure there.

Mr. Biden had already received Wall Street support early in the primary season. Both Jonathan Gray, the president of Blackstone, and Marc Lasry, the investor and big Democratic donor, co-hosted fund-raisers for Mr. Biden in Manhattan on Feb. 13. And Mr. Biden has taken in donations from the hedge fund manager Seth Klarman, Mr. Maitland and Stephen Scherr, the chief financial officer of Goldman Sachs.

Yet, on the campaign stump, Mr. Biden has invoked some of the same anti-elitist rhetoric that Mr. Sanders and Senator Elizabeth Warren have used to rile up Democratic voters. Speaking to a crowd in Los Angeles late Tuesday evening, as key Super Tuesday victories rolled in, Mr. Biden said, "Wall Street didn't build this country, you built this country." In his campaign literature, he has bemoaned the financial industry's putting profits over workers and the widening wealth gap in America.

The policy implications of a Biden administration for financial executives are not entirely clear. Mr. Biden has said he will shore up the Affordable Care Act, the landmark health-insurance reform act he helped enact during the Obama administration, which would presumably preserve the private-insurance system while enhancing government-subsidized coverage. He says he will reverse the Trump tax breaks and raise taxes on investment gains. Like Mr. Sanders, he has advocated for a $15 minimum wage. But he lacks the sort of detailed policy papers that Ms. Warren, a perennial critic of Wall Street, has generated, and his message is more focused on hope and solidarity than it is on attacking the current economic infrastructure.

Given all that, Mr. Cooperman said he would wait to hear more from Mr. Biden before committing to support him. "I'd like to see, hear more about what he's got to say," he said. But after watching the candidate surge past Mr. Sanders on Tuesday, he added, "hopefully the left wing of the Democratic Party understands they'll get murdered if they go with a left-wing candidate."

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Trump falsely claimed that Obama administration slowed down diagnostic testing, experts say - CNN

Posted: 04 Mar 2020 07:20 PM PST

"The Obama administration made a decision on testing that turned out to be very detrimental to what we're doing," Trump said Wednesday during on a meeting addressing the coronavirus outbreak. "And we undid that decision a few days ago so that the testing can take place in a much more rapid and accurate fashion."
An aide to Sen. Lamar Alexander, R-Tennessee, said the Obama administration made no such rule change. The aide, Taylor Haulsee, said the Obama administration did propose that the Food and Drug Administration have more oversight over approving diagnostic tests, but that did not go through.
White House seeks options to blunt economic fallout from coronavirus
"There has not yet been significant regulatory reform of diagnostics passed by Congress," Haulsee said.
Alexander served as chairman of the Senate Committee on Health, Education, Labor and Pensions while those changes were being considered during the Obama administration.
A policy expert at the Association of Public Health Laboratories agreed with Haulsee's assessment.
When asked about Trump's remarks, Peter Kyriacopolous, chief policy officer at the association, said: "We aren't sure what rule is being referenced."
He added that "there was an intense interest from FDA to pursue regulation of lab-developed tests during the Obama administration, but it never occurred. FDA did a lot of work on this, but there never was a final rule that came out of all that work."
The White House did not offer a comment when asked to explain why the President said it was an Obama-era decision.

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How Joe Biden Ran Up the Score in Virginia - The New York Times

Posted: 04 Mar 2020 06:50 PM PST

FALLS CHURCH, Va. — They were disaffected Republicans in affluent Washington suburbs. They were shipyard employees in Norfolk. And they were health care workers in Petersburg.

They all came together on Super Tuesday in an extraordinary surge to the polls in Virginia, propelling former Vice President Joseph R. Biden Jr. to an overwhelming victory in a state that just days earlier had seemed up for grabs. The triumph was part of a 10-state sweep for Mr. Biden that resurrected his presidential candidacy, and established him as the centrist Democrat who would go head-to-head with Senator Bernie Sanders of Vermont, the standard-bearer of the party's liberal wing.

In Virginia on Tuesday, it was no contest. Mr. Biden won with 53 percent of the vote, 30 percentage points more than Mr. Sanders. Voter turnout broke a state record for a presidential primary, and was especially high in suburban areas near Washington and near Richmond and Norfolk, as well as in regions with large African-American populations. Petersburg, a mostly-black city south of Richmond, went 75 percent for the former vice president.

The range of support suggested Mr. Biden had the potential to put together a broad coalition across categories of race, gender and age that could be a potent weapon in a race against President Trump.

Former Gov. Terry McAuliffe of Virginia attributed Democratic turnout in the state to deep disgust with the Trump presidency among Democrats and moderate Republicans.

"We have to give tremendous credit to Donald Trump," Mr. McAuliffe said. "He has been the single biggest driver to the Democratic Party of Virginia. There are a lot of like-minded Republicans who said, 'I can't vote for Trump but you got to give me somebody who we can vote for.' Biden was always at the top of that list."

That was the motivation for Laura Bligh, a 39-year-old personal trainer from Falls Church. She had planned to back Senator Elizabeth Warren of Massachusetts, but said she changed her mind on Monday and voted for Mr. Biden.

"My number one priority is to beat Trump and that seemed like the best chance to do it," Ms. Bligh said while waiting to pick up her daughter outside Shrevewood Elementary in Falls Church on Wednesday afternoon. "When there was still a large pool of candidates, minority options still had a chance."

Ms. Bligh said she considered her vote not just a repudiation of Mr. Trump, but an effort to block Mr. Sanders, who she predicted would lose in a bid against the president.

In Virginia, exit polls show that Mr. Biden dominated across gender, race and educational lines. He had support from men and women, white and black people, college graduates and non-graduates alike by double-digit margins over Mr. Sanders. In fact, in most of the demographic categories that Mr. Biden won, his share of the vote was larger than Mr. Sanders's and Ms. Warren's combined.

Even more significantly, Mr. Biden made major inroads among Mr. Sanders's core constituencies. For instance, Hispanic voters — who went overwhelmingly for Mr. Sanders in the Nevada caucuses — actually went for Mr. Biden by about 13 percentage points in the Virginia primary. And even among voters who described themselves as very liberal, Mr. Sanders won by only about 4 percentage points.

The only major demographic group that Mr. Sanders won in Virginia was young voters.

Mr. Biden's triumph here was clearly aided by his romp in the South Carolina primary on Saturday, and a string of endorsements from prominent Democratic politicians in Virginia — Senator Tim Kaine, Representative Bobby Scott and Mr. McAuliffe among them.

Exit polls also showed that Virginia voters coalesced around Mr. Biden's candidacy practically overnight. Nearly half said they made up their minds in the final days before Tuesday's contest, with two-thirds of those voters picking Mr. Biden.

The move to Mr. Biden was so swift and pronounced that his allies were claiming for their candidate the kind of surge in new voters that Mr. Sanders himself has promised but failed to deliver.

"The blue wave voters who flipped the House in 2018 and made Nancy Pelosi the speaker of the House, they really turned out in large numbers last night, and we overwhelmingly won their support," said Mayor Eric Garcetti of Los Angeles, a national co-chairman of Mr. Biden's campaign.

In a conference call with reporters, Mr. Garcetti cited turnout increases of 76 percent in three Virginia congressional districts that include parts of Norfolk and the suburbs of Washington and Richmond.

Over all, 1.3 million voters in Virginia went to the polls on Tuesday, surpassing the votes cast in 2016 by nearly 70 percent and, even more stunningly, breaking a previous Democratic record of 986,000 votes set in 2008, when Barack Obama was on the ballot.

Democratic turnout on Tuesday was up broadly compared with 2016 — with Texas up 49 percent, Tennessee up 38 percent, and Vermont and North Carolina both increasing by 16 percent. Several states that had switched from caucuses to primaries this year — Colorado, Maine, Minnesota and Utah — also saw heavy increases in participation.

Nowhere was the Democratic boom more pronounced than in the tony Northern Virginia suburbs of Washington. Here, among the million-dollar homes populated by lobbyists, corporate lawyers and employees of government contractors, Mr. Biden ran up huge margins over Mr. Sanders and the rest of the Democratic field while turnout in some areas nearly doubled from the party's 2016 primary.

The region, with its high population of educated professionals, was believed to be a good area for Ms. Warren and Pete Buttigieg, the former mayor of South Bend, Ind., before he ended his campaign on Sunday night.

Dan Helmer, who in November ousted the last remaining Fairfax County Republican in the Virginia General Assembly, said the rush of new voters helped catapult him into office. His victory, completing the Democratic sweep of the Washington suburbs, came after Mr. Trump alienated many of the federal workers and contractors.

"He has converted huge numbers of federal civil servants to Democrats," said Mr. Helmer, who switched his support from Mr. Buttigieg to Mr. Biden on Monday. "It's a pocketbook issue for Northern Virginia. Trump has just shook the earth in a way that it's going to take years for Republicans to recover."

The Falls Church precinct that voted at Shrevewood Elementary School, steps from a popular cycling trail that some commuters use to get to work in the capital, drew nearly twice as many voters on Tuesday as it did for the 2016 contest between Mr. Sanders and Hillary Clinton. Mrs. Clinton's margin over Mr. Sanders's in 2016 in that precinct was 99 votes; Mr. Biden beat Mr. Sanders by 250 votes.

For Sanders supporters, watching the turnout surge he had promised go to Mr. Biden served as a particularly bitter disappointment. They attributed the Vermont senator's weak showing in the suburbs to moderate Democrats' coalescing around Mr. Biden faster than progressives did behind Mr. Sanders — though in Fairfax County, Michael R. Bloomberg, the former mayor of New York, won more votes than did Ms. Warren. They also cited the rapid nationalization of a race they had hoped to win through on-the-ground organizing.

"Bernie Sanders, he reached out to Latino voters, Muslims, Arab-American — these were constituencies that he had a significant amount of support here," said Yasmine Taeb, a Sanders-aligned Democratic National Committee member from Falls Church. "While there was an increase in the number of young voters and minority voters, it still didn't match up to the older white voters that you traditionally see voting in primaries."

Jonathan Sokolow, a labor lawyer who is co-chairman of the Sanders campaign in Virginia, said the results in Virginia and elsewhere were not an indication that Mr. Sanders did not have wide support in the party.

"I don't think it's correct to say that we don't have majority support," Mr. Sokolow said. "We're fighting to see who is the best candidate to defeat Donald Trump."

But across the Virginia suburbs, Mr. Sanders's argument fell flat in the face of Mr. Biden's late strength.

Marcus Simon, a Democrat who represents Falls Church in the Virginia House of Delegates, said he voted early on Saturday for Ms. Warren. By Tuesday, he had changed his mind, and encouraged his wife to vote for Mr. Biden.

"Electability is something everybody is thinking about and who has the best chance of defeating Trump," he said. "The idea of risking the nomination to somebody like Bernie Sanders, the concern would be that he wouldn't have the broad appeal to defeat Donald Trump."

Tucker Martin, who a decade ago was a top aide to former Gov. Bob McDonnell, Republican of Virginia, appears to fit into this category. Mr. Martin considers himself a Republican, but on Super Tuesday, he cast a ballot for Mr. Biden.

"I am a Never Trumper and I'm out of partisan politics," Mr. Martin said on Wednesday. "I think for many voters in Virginia, Joe Biden may represent a safe place to go to register their dislike for the current occupant of the White House."

Maggie Astor and Thomas Kaplan contributed reporting from New York.

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エンタメ関連各社が声明文発表「感染が広がらないようにつとめる」――新型コロナによるイベント中止を受けて - 紀伊民報

Posted: 04 Mar 2020 06:44 PM PST

　一般社団法人日本音楽事業者協会、一般社団法人日本音楽制作者連盟、一般社団法人コンサートプロモーターズ協会のエンターテインメント関連３団体および主要コンサート制作会社らは4日、新型コロナウイルス感染拡大の防止を呼びかけるため、「エンターテインメントを愛する皆さんへ」と題する声明文を発表した。

【写真】池上彰氏が"新型コロナ"東京五輪の影響を分析

　スポーツや音楽ライブ、劇場公演などのイベントの実施について政府は、「今後2週間は中止や延期、規模縮小するよう要請する」と表明した。このことを受け、エンターテインメント関係各社は現在、予定していた数多くのイベントの中止・延期の措置を継続している。

　こうした現状に対し、声明文では「開催に向けて準備してきた苦労や、開催することで得られる充実感や達成感、そして何よりもオーディエンスの皆さんと分かち合えるかけがえのない喜びなど、経済的な損失以上に、その代償は大きなものがあります」としつつも、「エンターテインメントが再び多くの皆さんに強力な影響を与えることができるよう、細やかな知識や情報を、率先して広げていきたい」と、改めてエンターテインメント業界側の姿勢を示した。

　そのうえで、まずはエンターテインメント業界側が実施可能な感染防止策を徹底する一方で「少しでも沢山の皆さんが感染拡大防止の知識を増やし、事態の収束への意識を強く持ち、新型コロナウイルスに1日も早く打ち克ち、その結果、共に楽しいエンターテインメント空間を描けることを心より願っています」とし、政府が発表している「手洗い」「マスクの着用を含む咳エチケット」等の感染症予防の手引きを添えて、感染対策を呼び掛けた。

■声明文（全文）

エンターテインメントを愛する皆さんへ

先般のイベント中止、もしくは延期の要請を受け、コンサート等のエンターテインメントを中断する判断や、敢行する判断に対する様々な批判や賛同がある中で、我々は大多数の公演を中止、もしくは延期することに致しました。

何よりも皆さんの健康が優先されることが大事という、強い気持ちを持って判断した次第です。

一方で、開催に向けて準備してきた苦労や、開催することで得られる充実感や達成感、そして何よりもオーディエンスの皆さんと分かち合えるかけがえのない喜びなど、経済的な損失以上に、その代償は大きなものがあります。

だからこそ、今回の新型コロナウイルスに対して今一度正しい知識と関心を持っていただき、これ以上の感染が広がらないようにつとめる事を、率先して訴えかけます。

エンターテインメントが再び多くの皆さんに大きな力を与えることができるよう、細やかな知識や情報を、率先して広げていきたいと思います。

我々も、開催に向け実施可能な感染防止対策を行いますので、少しでも沢山の皆さんが感染拡大防止の知識を増やし、事態の収束への意識を強く持ち、新型コロナウイルスに1日も早く打ち克ち、その結果、共に楽しいエンターテインメント空間を描けることを心より願っています。

エンターテインメントを愛する皆さんと共に。

#春は必ず来る

2020年3月4日

一般社団法人日本音楽事業者協会
一般社団法人日本音楽制作者連盟
一般社団法人コンサートプロモーターズ協会

ジャニーズ、グループ主催公演の延期と休演を発表　新型コロナウイルス感染状況など鑑み
AKB48、モー娘、ももクロ競演番組が無観客収録に　NHKの新型コロナ感染拡大防止策で
嵐、北京公演中止を発表　新型コロナウィルスの感染拡大受け
WANIMA、福井・愛媛公演中止を発表「コロナウイルス予防・拡散防止のため」
電通、本社ビル勤務全従業員に在宅リモートワーク実施　50代男性がコロナウイルス陽性

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March 04, 2020 at 01:00AM
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エンタメ関連各社が声明文発表「感染が広がらないようにつとめる」――新型コロナによるイベント中止を受けて - 紀伊民報
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Transfer news and rumours LIVE: Man Utd hopeful of deals for both Grealish and Sancho - Goal.com

Posted: 04 Mar 2020 06:38 PM PST

The Dortmund prodigy's price is rising with his excellent performances

Borussia Dortmund star Jadon Sancho's hot form will force Man Utd, Chelsea or Liverpool to break the Premier League's transfer record to sign the teenager.

According to BILD, the 19-year-old's value has risen to €140 million (£122m/$156m) with his extraordinary performances for Dortmund amounting to 17 goals and 18 assists in 33 matches across all competitions.

English giants United, Chelsea and Liverpool have been linked strongly with the Three Lions international and to sign Sancho, they will be forced to break the Red Devils' Premier League record signing of £89m for Paul Pogba.

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After Losses on Super Tuesday, Elizabeth Warren Reviews Her Options - The New York Times

Posted: 04 Mar 2020 06:20 PM PST

DETROIT — Senator Elizabeth Warren faced an uncertain path forward on Wednesday after a Super Tuesday performance that fell below her campaign's already lowered expectations, with her chances at the Democratic presidential nomination now a mathematical anomaly.

Ms. Warren finished third in her home state, Massachusetts, and failed to crack the top two in any contest, leaving any possibility that she could win the nomination reliant on party chaos and not her own electoral prowess.

Her campaign manager, Roger Lau, wrote in an email to staff members on Wednesday morning that Ms. Warren was assessing her options.

Mr. Lau said the team was "obviously disappointed," writing, "We fell well short of viability goals and projections."

As for the next steps, he said Ms. Warren was "going to take time right now to think through the right way to continue this fight."

He added, "This decision is in her hands, and it's important that she has the time and space to consider what comes next."

Earlier on Wednesday, Michael R. Bloomberg dropped out of the race and endorsed Joseph R. Biden Jr., fully uniting the moderate wing of the Democratic Party behind Mr. Biden and leaving progressives split between Mr. Sanders and Ms. Warren — though, as polling has suggested, many voters do not make their decisions strictly based on ideology.

Mr. Sanders, speaking to supporters in Vermont on Wednesday afternoon about his own disappointment with the Super Tuesday results, said that he had spoken with Ms. Warren several hours earlier by phone and that she had told him she was "assessing her campaign" but had not made any decisions yet.

"She has not made any decisions as of this point," Mr. Sanders said, "and it is important, I think, for all of us — certainly me, who has known Elizabeth Warren for many, many years — to respect the time and the space that she needs to make her decision."

Maurice Mitchell, the national political director for the Working Families Party, which has endorsed Ms. Warren, said he was "pleased" the two senators "are now speaking."

"They're both giants of the progressive movement, and we are hopeful that they can figure out how to work together to ensure a progressive nominee and a progressive agenda in Washington," Mr. Mitchell said.

The results on Tuesday represented a crushing blow to Ms. Warren's hopes. Last month, Mr. Lau had predicted that she would finish in the top two in eight of the 14 Super Tuesday states, ensuring supporters that, if only they were patient, she still had a path to the nomination regardless of the results in Iowa and New Hampshire.

Now Super Tuesday has come and gone, and Ms. Warren was the only one of the four leading candidates to not score an outright victory — even in Massachusetts, where she fell behind Mr. Biden, who won, and Mr. Sanders.

Ms. Warren is now far behind the race's new delegate leader, Mr. Biden, and Mr. Sanders, who have emerged as the clear front-runners.

In an email to supporters on Tuesday night, Ms. Warren's campaign had ignored the results, trying to cast the race as a long haul with a lack of clarity all around.

"Delegates have to be counted and allocated by congressional district or State Senate district, and that process takes time," the email said. "But here's the bottom line: There are six more primaries just one week away, and we need your help to keep up the momentum."

But outside that email to supporters, Ms. Warren's official campaign apparatus was silent Tuesday evening — making no mention of being shut out of delegates in Texas and California, or her third-place finish in Massachusetts.

Communications staff members, organizing aides and prominent supporters also avoided questions about her performance, with some reaffirming on Twitter that they remained a "Warren Democrat."

Amid her campaign's silence, there were mounting calls for her to exit the race — and speculation, if she were to drop out, about whom she might endorse.

She told one supporter Wednesday that she was not going to act quickly and that a decision would most likely not be for at least another day. Some urged her to take her time, while others were pressing her to use whatever leverage she had now and endorse Mr. Biden, according to Democrats who have spoken to her.

Representative Ilhan Omar of Minnesota, a high-profile surrogate for Mr. Sanders, suggested on Twitter that Ms. Warren's presence in the race was stopping progressives from being able to overtake the party's moderate wing.

"Imagine if the progressives consolidated last night like the moderates consolidated, who would have won?" Ms. Omar asked. "That's what we should be analyzing."

Progressive organizations also weighed in. Left-wing advocacy groups that had endorsed Mr. Sanders but were previously wary of criticizing Ms. Warren made clear on Tuesday evening that they believed the primary had become a choice between two candidates: Mr. Biden and Mr. Sanders — not Ms. Warren.

"We need energy and enthusiasm to beat Donald Trump," said Natalia Salgado, the political director for one group that backed Mr. Sanders, the Center for Popular Democracy Action. "No candidate is inspiring voters in the same way as Bernie Sanders is."

For Ms. Warren, the disappointing results came even after she had some fortunate breaks. Aides believed that she would benefit from other candidates' dropping out the race, and that the departures of former Mayor Pete Buttigieg of South Bend, Ind., and Senator Amy Klobuchar of Minnesota could help her reach the 15 percent threshold for earning delegates in key states like California and Texas.

She was above 15 percent in only a few California districts, mostly the most liberal and well-educated enclaves: three Bay Area districts (based in San Francisco, Berkeley and Silicon Valley) and two in the Los Angeles area that included West Hollywood and Santa Monica.

And Ms. Warren also had help in recent weeks from the infusion of money by a huge super PAC — the biggest in the race — that plunged nearly $15 million into Nevada, South Carolina and the Super Tuesday states.

Regardless, Ms. Warren had picked up so few clear or decisive delegate wins on the Eastern Seaboard and across the South that, as the polls closed in California, she was still behind Mr. Buttigieg, who had dropped out before Super Tuesday, in the live delegate count that NBC News had maintained. (She eventually passed him.)

Even so, Ms. Warren's campaign had known that Tuesday would not be an uplifting night. While Mr. Sanders and Mr. Biden scheduled major events for after the polls had closed, hoping to revel in the sweet taste of victory, Ms. Warren did a lower-key event in Michigan earlier in the evening.

More than 2,000 people attended, according to the campaign, cheering hardest when Ms. Warren asked them to disregard pundit worries and "cast a vote that will make you proud, cast a vote from your heart, and cast a vote for a person who you think will make the best president."

She did not stay for a selfie line, a tradition that was a fixture of her campaign but became less frequent as the imperatives of travel intensified.

Her opening speaker compared her campaign to a phoenix, the creature in Greek folklore that obtains new life by arising from the ashes of its predecessor.

"Elizabeth Warren is a lot like Detroit," said the speaker, Stephanie Chang, a state senator in Michigan. "She is scrappy. She never gives up. She hopes for better things, and like the phoenix, she will rise!"

Astead W. Herndon reported from Detroit, and Shane Goldmacher from New York. Sydney Ember contributed reporting from Burlington, Vt., and Jonathan Martin from New York.

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Corbin Steele's lone goal lifts Chelsea hockey past Livonia Churchill in D3 regional final - MLive.com

Posted: 04 Mar 2020 05:38 PM PST

Jenna Kieser | jkieser@mlive.com

Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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ANN ARBOR – Chelsea's hockey team defeated Livonia Churchill 1-0 to claim its second regional championship in three seasons.

The Bulldogs withstood multiple shots on net by Churchill and took advantage of a scoring opportunity when Devin McIntyre found Corbin Steele, who slapped in the lone goal with six minutes remaining in the third period.

Chelsea was able to hold off Churchill down the stretch to improve to 14-11-2 on the season and move on to the Division 3 quarterfinal, where it will take on the winner of Riverview and Riverview Gabriel Richard Saturday at the Dearborn Ice Skating Center.

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Chelsea's Gabriel Vowles (16) and Livonia Churchill's Cameron Greener (16) fall to the ground during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players lift up the D3 regional championship trophy after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Corbin Steele (11) brings the puck down the ice during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill's Keith Peterson (15) watches the regional finals from the bench at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Fans watch from the stands as Chelsea takes the ice for the second period of the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Wesley Hansen (22) and Livonia Churchill's Dominic Krupinski (7) collide during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Jenna Kieser | jkieser@mlive.com

Chelsea coaches celebrate a goal from the bench during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020. The goal was the only goal of the game and won the team the D3 regional title.

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Chelsea's Jack Capper (14) hits the puck down the ice during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players pose with the D3 regional championship trophy after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill's Michael Mancina (22) is pinned to the board by Chelsea's Gabriel Vowles (16) during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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A fan knuckle touches the referees as they take the ice for the third period of the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Wesley Hansen (22) and Jack Capper (14) climb over the boards to go back in the game during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Corbin Steele (11) and Livonia Churchill's Cameron Greener (16) go for the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Corbin Steele (11) stops for a water break during a timeout in the last minutes of the game at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill's Dominic Krupinski (7) skates past Chelsea players during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Tyler Valik (21) skates the puck around Livonia Churchill's Donovan Young (14) during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players listen to head coach Nick Vetter during a timeout in the final minutes of the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea and Livonia Churchill players keep their eyes on the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill fans hold homemade signs at the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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A Chelsea player and Livonia Churchill's Michael Mancina (22) search for the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Fans watch from the stands as Chelsea and Livonia Churchill compete in the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Gabriel Vowles (16) keeps control of the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill players take the ice for the D3 regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Jack Capper (14) keeps an eye on the puck after a face off during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill's Ryan McCoy (13) falls to the ground during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate a goal during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Gabriel Vowles (16) and Livonia Churchill's Ryan McCoy (13) go for the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill goalie Bryant Riley (33) leans against the net while watching the regional final game at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Wesley Hansen (22) and Livonia Churchill's Ryan Darling (11) go for the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Devin McIntyre (19) and Livonia Churchill's Justin Haydu (6) keep an eye on the puck during a face off during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill's Jimmy Targosz (3) weaves around Chelsea players during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Young fans climb on the stands between periods of the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea's Wesley Hansen (22) and Livonia Churchill's Michael Mancina (22) and Justin Haydu (6) fight for possesion of the puck during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Livonia Churchill's Keith Peterson (15) attempts to take the puck from Chelsea's Tyler Valik (21) during the regional finals at the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Chelsea players celebrate winning the D3 regional championship trophy in the locker room after winning the game 1-0 over Livonia Churchill the Ann Arbor Ice Cube on Wednesday, March 4, 2020.

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Kylian Mbappe Scores Outrageous Solo Goal for PSG in Coupe de France Semifinal - Sports Illustrated

Posted: 04 Mar 2020 05:38 PM PST

Kylian Mbappe in the open field is a scary proposition, something Lyon found out first-hand on Wednesday.

Mbappe acted as a one-man counterattack in their Coupe de France semifinal, giving PSG a two-goal cushion in the second half of a 5-1 win with his second of three goals in the game. He collected possession about halfway inside his own half before taking off, putting on the afterburners to avoid one defender, juking another and then finishing from inside the box.

PSG actually trailed 11 minutes in on Martin Terrier's goal, before Mbappe struck three minutes later by getting the final touch on Layvin Kurzawa's header off a corner that appeared to be going in anyway.

Neymar gave PSG the lead in the 64th minute on a penalty kick before Mbappe worked his magic six minutes later for some insurance. Pablo Sarabia put the match to bed with PSG's fourth in the 81st minute, before Mbappe capped his hat trick in stoppage time.

Wednesday marked Mbappe's second multi-goal game in a row after his double over the weekend in Ligue 1, and he now has 30 goals in all competitions this season.

PSG will play the winner of Thursday's Rennes vs. Saint Etienne semifinal for the trophy. PSG fell to Rennes in last season's final, ending a streak of four straight titles.

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Trump’s Economic Cheerleading Is Suddenly Tested - The New York Times

Posted: 04 Mar 2020 05:20 PM PST

WASHINGTON — One of President Trump's greatest strengths in his presidential campaign is his economic salesmanship: He has convinced a devoted share of Americans that his leadership has made the U.S. economy bulletproof and markets would crash if he were defeated in November.

The president's ability to set the economic narrative is buoyed by an 11-year economic expansion, with rising wages and unemployment at a 50-year low. That strength presents a challenge for the Democrats hoping to unseat him in November — even though Mr. Trump's cheerleading is often overstated and parts of the economy are slowing, including manufacturing.

That helps explain why Mr. Trump has played down economic damage from the coronavirus and dismissed the stock market plunge: They threaten to undermine the most effective story he tells about his presidency.

"The country is in great shape, the market is in great shape," Mr. Trump told reporters outside the White House on Tuesday, as stocks tumbled after the Federal Reserve's rate cut. The S&P 500 ended the day down about 2.8 percent.

Mr. Trump has blamed any growth hiccups on external events, like the Democrats running for president, troubles at the aerospace giant Boeing and the Federal Reserve. He has praised consumers and the economy's strength, even as forecasters warn that the virus could dampen growth at least temporarily this year.

On Monday, Mr. Trump suggested on Twitter that House Democrats pass a one-year cut in payroll taxes, a form of fiscal stimulus often aimed at boosting consumer spending at times of economic weakness. Yet the president did not concede any economic trouble.

"This is an incredible time for our nation," he wrote on Twitter. "Jobs are booming, incomes are soaring, poverty is plummeting, confidence is surging."

That kind of rhetoric has served Mr. Trump well during his first three years in office, helping him win credit — at least among supporters — for what remains a strong economy. But experts warn that it has also helped widen a partisan divide on the economy, which may make it more difficult for Mr. Trump to reassure the public in the event of a crisis, like the spread of coronavirus.

"The aura of the office, where people might have deferred to the message about the state of the economy, I think today that's gone," said Shanto Iyengar, a political scientist at Stanford University. "It's gone because of the hyper-partisanship."

Americans who tune into Mr. Trump's messages are far more likely to echo his language on the economy and are more optimistic about it, according to a new nationwide poll conducted last month for The New York Times by the online research firm SurveyMonkey.

Respondents were asked to choose from a list of terms that could describe the state of the economy. Those who said they regularly watched or followed news coverage of Mr. Trump's speeches were about twice as likely to describe the economy as "booming" or the "best economy ever," and they were far more likely to express confidence in the economy and give Mr. Trump credit for its condition.

The split persisted among Democrats, independents and even among Republicans. More than eight in 10 Republican voters who regularly follow Mr. Trump's speeches say Mr. Trump deserves "a lot" of credit for the economy. Only about one in three Republicans who do not regularly follow the speeches said the same.

Will Hicks, a 32-year-old survey respondent in the oil field town of Roosevelt, Utah, said Mr. Trump's election provided an immediate jolt to people in his area. He credits Mr. Trump and his boosterism for much of that, even though he said the president's claims were often overstated.

"His viewpoint is all positive, that we're going to get greater, we're going to improve our economy," Mr. Hicks said. "I think that push forward, even though it might be a little bit of an exaggeration, has helped the economy."

Some economists agree. Robert Shiller, a Nobel Prize-winning economist at Yale, has described Mr. Trump's cheerleading as the driving force behind the continuation of the now-record economic expansion over the last three years.

In a presentation earlier this year — in which he compared Mr. Trump's economic messaging to the spread of a pandemic virus — Mr. Shiller reported than 62 percent of newspaper articles that mentioned a "strong economy" from 2017 to 2019 also mentioned Mr. Trump. That's more than twice the rate of association that President Bill Clinton enjoyed during a stronger economy in the late 1990s.

Mr. Trump "is our first motivational-speaker president," Mr. Shiller said.

Mr. Trump made grand economic promises a centerpiece of his 2016 campaign, and he reveled in the stock-market surge that followed his victory. Even before he took office, consumer and small-business confidence levels soared, driven largely by Mr. Trump's Republican supporters. Both measures have remained elevated throughout his term, with sentiment among Republican consumers hitting a record high in February in a long-running survey from the University of Michigan.

But on several measures, the economy has fallen short of the president's promises. It grew 2.3 percent last year, well below the forecasts of Mr. Trump's economic team, as the president's trade war chilled business investment. Economic growth in the first three years of President Barack Obama's second term was nearly identical to that of Mr. Trump's first three years. Median wage growth is no higher today than it was in October 2016, and it remains well below the levels of the late 1990s.

Small business owners' expectations for capital investment now are essentially unchanged from before the 2016 election. Among consumers, the postelection rise in confidence has failed to translate into a sustained improvement in spending, shattering a longstanding relationship. After Mr. Trump's election, said Ian Shepherdson, chief economist at Pantheon Macroeconomics, "a gap opened up between the spending and confidence numbers, and that gap has persisted."

Feeling Good, But Not Acting Like It

President Trump's election led to a surge in consumer confidence, at least among Republicans. But that hasn't translated into stronger spending.

CONSUMER CONFIDENCE

CONSUMER SPENDING

University of Michigan

Index of Consumer Sentiment

Year-over-year percentage change in

inflation-adjusted consumer spending

120

+6

%

NOV. '16

NOV. '16

Trump elected

Trump elected

+4

100

+2

80

0

60

–2

40

–4

20

–6

12-MONTH ROLLING AVERAGE

12-MONTH ROLLING AVERAGE

0

–8

'00

'05

'10

'15

'00

'05

'10

'15

Consumer confidence

Consumer spending

University of Michigan

Index of Consumer

Sentiment

Year-over-year percentage

change in inflation-adjusted

consumer spending

120

+6

%

NOV. '16

NOV. '16

Trump elected

Trump elected

+4

100

+2

80

0

60

–2

40

–4

20

–6

12-month rolling avg.

12-month rolling avg.

0

–8

'00

'05

'10

'15

'00

'05

'10

'15

Sources: Bureau of Economic Analysis (consumer spending); University of Michigan (consumer confidence).

By The New York Times

The confidence gap is driven entirely by asymmetric partisanship, powered by a surge in Republican sentiment. When Mr. Obama was in office, surveys consistently showed that Democrats felt better about the economy than Republicans. When Mr. Trump was elected, the pattern reversed almost overnight, though the surge in Republican optimism was larger than the drop-off among Democrats.

Masha Krupenkin, a Boston College political scientist, said Republicans show bigger swings in economic confidence, and exhibit a similar pattern in surveys that ask about trust in government, depending on which party controls the White House, compared with Democrats.

The Times survey showed Democrats were less optimistic about the economy and less willing to credit Mr. Trump for it than Republicans were. Confidence among independent voters has fallen to a level between that of Democrats and Republicans, but closer to Democrats', during Mr. Trump's term. (Optimism among independents surged in January, but came back to earth in February, according to the Times survey.)

Douglas Prasher, a Democratic voter in Prescott Valley, Ariz., said he didn't buy claims that the economy was doing well — and he didn't think Mr. Trump deserved credit if it was. Mr. Prasher, 68, recently retired to Arizona from San Diego, which he said had become unaffordable because of sky-high housing costs. His new home state is cheaper, but he has still had to take on a part-time job to make ends meet. He carries hundreds of thousands of dollars in student loan debt from helping his daughters go to college.

"As far as I'm concerned, people are suffering," Mr. Prasher said. The supposedly strong economy, he said, "seems to only help the wealthy, which we're not in that category."

That rhetoric echoes the message of Democratic candidates running for the White House, who frequently claim that the economy is "rigged" or only works for the very rich. Democrats in the Times survey were much more likely to use that phrasing to describe the economy than Mr. Trump's preferred terms. So were independents, who were six times as likely to call the economy "rigged" or badly broken" as to say it was the "best economy ever."

Mr. Trump's aggressive claims — that it is not just strong but "the best ever" — may make him less credible to people not already inclined to support him, said Michele Claibourn, a political scientist at the University of Virginia. "It's easier to resist as information if it feels exaggerated," she said.

Ms. Claibourn said presidents have historically tried to distinguish between when they were speaking as party leaders and when they were addressing the public in their official, less partisan, capacity. Those lines have blurred over the decades. Under Mr. Trump, the distinction has disappeared almost entirely, she said.

Jim Tankersley reported from Washington, and Ben Casselman from New York.

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Acclaimed actress Alexis J. Roston stars in Ella Fitzgerald tribute - Chicago Tribune

Posted: 04 Mar 2020 05:19 PM PST

Unfortunately, our website is currently unavailable in most European countries. We are engaged on the issue and committed to looking at options that support our full range of digital offerings to the EU market. We continue to identify technical compliance solutions that will provide all readers with our award-winning journalism.

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Ducks' Carter Rowney: Registers seventh goal - CBSSports.com

Posted: 04 Mar 2020 05:08 PM PST

Rowney posted a goal and six hits in Tuesday's 6-2 loss to the Blackhawks.

Rowney toils in the bottom six, mainly on the fourth line, and averages 12:43 per game, so it's not surprising that he'll struggle to achieve a new career high in points (21). However, the hard-nosed winger notched his seventh goal to tie the career best he set last year, and he's now surpassed 180 hits for the first time in his career.

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Solskjaer issues goal warning to 'old dog' Rooney ahead of Man Utd's trip to Derby - Goal.com

Posted: 04 Mar 2020 05:08 PM PST

Ole Gunnar Solskjaer quipped that any goals "old dog" Wayne Rooney scores for Derby County against Manchester United on Thursday will be chalked off his record tally.

Rooney is set to captain Derby against his former club in an FA Cup fifth-round tie at Pride Park.

The former England captain scored a record 253 goals in 559 games for United before returning to Everton in 2017 and joined the Rams as a player-coach in January after a spell in MLS with DC United.

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Solskjaer knows his side must be wary of his former team-mate Rooney, who is his son Noah's favourite player, and joked the 34-year-old should have a good reason to avoid finding the back of the net.

The United manager told a press conference: "He gave absolutely everything for this club and we all appreciate what he's done, but any goal he scores against us will be chalked off his goal tally, so he doesn't want to score too many.

"Wayne will show what he can do, he wants to prove there is still fight in an old dog. We've got to be on our toes, don't give him any space in and around the box, or in midfield, because you can see his Scholesy-like passing range."

Solskjaer went on to dismiss any suggestion that Rooney's contribution to United's cause has been undervalued, adding: "He's been a fantastic player for Man United. And he played here for so long. He's the club's top scorer, he won so many trophies.

"For me, I don't think the fans underrate him, I think everyone really, really appreciates what he did for us, he's still very welcome, he's still around the place, he still comes to Old Trafford, so he will get a good reception from our away fans in that game."

The Norwegian head coach added on Rooney's ambitions to move into management himself when he finally hangs up his boots: "I think he just wants to stay in the game, as many of us do, and he's a great person, he makes everyone feel good – whether you are 80 or 10.

"He's even my Noah's favourite player, not just because what he did as a player, but the kind of human being he was in and around the place.

"I'm sure when he becomes a manager he'll affect his players. When he's coaching, he's so enthusiastic about the game and he knows the game."

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NCT 127 Returns With Powerful New Release ‘Kick It’, Hits Twitter Trends Worldwide - Sputnik International

Posted: 04 Mar 2020 04:50 PM PST

NCT 127 attending a fan meeting in Bangkok in September 2017
© Photo : Bugaboo.TV

The K-Pop boy band comeback song is a Bruce Lee-style knock out.

The 10-member group NCT 127 dropped their new MV today for the upcoming album "Neo Zone". The band's second full-length album, which will include 13 tracks, will be released on 6 March.

The album's lead track – 'Kick It' – also goes by the Korean title 영웅, which is translated as "Hero"; and the members of NCT 127 show off their flaming charisma while transforming into heroes in the music video, demonstrating some classic Bruce Lee moves while also mentioning him in the lyrics.

​The whole comeback was promising and was a nostalgic homage to old movies and games from the start, as when it was first announced on 12 February, the timeline was full of photo and video content.

​The band previously presented different video teasers for every song of the upcoming album with a set of pictures for every member, showing off the wide range of songs they are able to perform - from touching ballads to aggressive hip-hop.

Shortly after the music video release, supportive hashtags such as #KickItWithNCT127 and #Bruce Lee were trending worldwide both in English and the Korean segment of Twitter, as NCTzen (the name of the fandom) shared caps and gifs from the MV admiring their idols.

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花とゆめ・LaLa・ザ花・LaLaDX10冊分を無料公開、休校要請受けて - ナタリー

Posted: 04 Mar 2020 08:07 AM PST

花とゆめ・LaLa・ザ花・LaLaDX10冊分を無料公開、休校要請受けて - ナタリー

花とゆめ、LaLa、ザ花とゆめ、LaLaDX（すべて白泉社）の電子版合計10冊分が、本日3月5日から4月2日まで白泉社e-net!で無料公開されている。

これは新型コロナウイルスの感染拡大による、全国の小・中・高校への休校要請を受けてのもの。白泉社では全国の学生に寄り添い、心の支えになりたいとの思いから無料公開を決めた。対象となるのは、花とゆめ1～5号、ザ花とゆめ3月1日号、LaLa1～3月号、LaLaDX3月号。この機会に、少女マンガ雑誌をじっくりと読んでみては。

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2020-03-04 15:00:00Z
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休校中の子どもにおうち内通貨「ママペイ」 お母さん自作のお手伝い促進アイデアが面白い - ねとらぼ

Posted: 04 Mar 2020 06:06 AM PST

休校中の子どもにおうち内通貨「ママペイ」 お母さん自作のお手伝い促進アイデアが面白い - ねとらぼ

　Twitterで「#休校中におすすめの過ごし方」（関連記事）として投稿された、お母さんによる子どもをやる気にさせるアイデア「ママペイ」が、子どもの生活リズムの安定や自主的なお手伝いを促してくれそうなステキな発想です。

　簡単に説明すると、ポイント制のお手伝いチェックシートのようなもので、各目標をクリアするとポイントの代わりに架空通貨（単位：ペイ）がもらえます。そして貯めたペイで「好きなおやつ」や「ゲームの時間」などを買うというシステムです。お仕事体験で専用通貨がもらえる「キッザニア」みたいで楽しそう！


休校中におすすめの過ごし方 子ども ママペイ 生活習慣 キッザニア 工夫 アイデア守ってほしい生活習慣やお手伝いを「ママペイ」がもらえる"クリア目標"として一覧に

　クリア目標一覧には、お手伝い以外にも「朝7時におきる」や「夜8時までにふろ入る」といった生活習慣も入っており、自動的に正しい生活習慣を定着させることにつながります。ちなみに一覧表は額に入れ、通貨の"ペイ"は名刺サイズのメッセージカードに手描きで作成。すべて100円ショップのアイテムで作っています。


休校中におすすめの過ごし方 子ども ママペイ 生活習慣 キッザニア 工夫 アイデアママペイ通貨は100円ショップのメッセージカードで作成

　小学4年生と小学1年生の息子さんを持つお母さんのri（@springda321）さんが投稿。実際に兄弟2人ともハマってくれたそうで、riさんも「色々言わなくても子どもみずから行動できるようになって拍子抜けしてます」と、ビックリするほど日々の悩みが減ったようです。


休校中におすすめの過ごし方 子ども ママペイ 生活習慣 キッザニア 工夫 アイデアすごい楽しそう！

　政府の要請により、少し早めの春休みとなってしまった子どもたち。特に今は暇を持て余すことも多そうですが、自作の「ママペイ」を導入することで、お子さんの楽しみを増やすと同時に、決まった時間に寝起きするなどの健康的な生活が保たれそうです。

画像提供：@springda321さん

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2020-03-04 12:15:00Z
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