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Smad2 Mediates Transforming Growth Factor-β Induction of Endothelial Nitric Oxide Synthase Expression
Transforming growth factor-β (TGF-β) increases expression of endothelial nitric oxide synthase (eNOS), although the precise mechanism by which it does so is unclear. We report that Smad2, a transcription factor activated by TGF-β, mediates TGF-β induction of eNOS in endothelial cells. TGF-β induces Smad2 translocation from cytoplasm to nucleus, where it directly interacts with a specific region of the eNOS promoter. Overexpression of Smad2 increases basal levels of eNOS, and further increases TGF-β stimulation of eNOS expression. Ectopic expression of Smurf, an antagonizer of Smad2, decreases Smad2 expression and blocks TGF-β induction of eNOS. Because Smad2 can interact with a variety of transcription factors, coactivators, and corepressors, Smad2 may thus act as an integrator of multiple signals in the regulation of eNOS expression.
Although endothelial nitric oxide synthase (eNOS or NOS3) was originally described as a constitutive NOS, subsequent work showed that eNOS expression is regulated by a variety of physiological and pathophysiological factors.1–6⇓⇓⇓⇓⇓ Expression of eNOS is increased by exercise, shear stress, hypoxia, hyperthyroidism, cirrhosis, and endothelial cell proliferation.7–10⇓⇓⇓ Various mediators can also increase eNOS expression, including estrogens, angiotensin II, glucose, oxidized linoleic acid, lysophosphatidylcholine, hydrogen peroxide, and transforming growth factor-β.1–6⇓⇓⇓⇓⇓ Expression of eNOS is decreased by hypoxia, inflammation, and pulmonary hypertension.11–14⇓⇓⇓ Mediators that decrease eNOS expression include oxidized LDL, lipopolysaccharide, and tumor necrosis factor-α.
Regulation of eNOS Expression
The level of eNOS expression is determined by multiple mechanisms, including posttranscriptional regulation15–18⇓⇓⇓ and posttranslational regulation.19–36⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓⇓ Expression of eNOS is also regulated at the transcriptional level. The 5′-flanking region of the eNOS gene lacks a TATA box, but contains elements that mediate constitutive expression of genes in endothelial cells, including Sp1 sites and GATA.37–42⇓⇓⇓⇓⇓ The 5′-flanking region also contains many putative sites for further transcriptional regulation of eNOS. However, only a few of these sites have been formally shown to regulate eNOS transcription, including PEA3 and AP-1 binding sites.43,44⇓ Finally, a region of the eNOS promoter extending from −1269 and −935 mediates TGF-β induction of eNOS transcription.45
TGF-β and Vascular Pathophysiology
TGF-β belongs to a superfamily of dimeric growth factors, including bone morphogenetic proteins and activins.46–48⇓⇓ TGF-β regulates diverse biological processes, including cell proliferation, differentiation, and migration. TGF-β also regulates cellular functions within the cardiovascular system, including angiogenesis, fibrosis, and production of extracellular matrix.
TGF-β Signal Transduction and Smads
After its secretion and activation, TGF-β forms a complex with type I and type II TGF-β receptors on target cells. Once bound to TGF-β, the type II TGF-β receptor phosphorylates the type I receptor, and the type I receptor in turn phosphorylates transcription factors known as Smads. Smad family members mediate TGF-β signal transduction, and include receptor Smads (Smad2 and Smad3) that associate with and are activated by TGF-β receptors, common Smads (Smad4) that interact with receptor Smads, and inhibitory Smads (Smad6 and Smad7) that block receptor Smad signaling.46,49–51⇓⇓⇓ The TGF-β type I receptor phosphorylates Smad2, which then disassociates from the receptor, binds to Smad4, and enters the nucleus. This Smad complex then interacts with other transcription factors, coactivators, and corepressors to modulate gene transcription.52
TGF-β Regulates eNOS
TGF-β regulates eNOS through pathways that are partially understood.45,53⇓ Harrison and colleagues45 previously showed that TGF-β regulates eNOS expression in part by activating NF-1 to bind to the eNOS promoter region −1269 and −935. We hypothesized that the Smad signal transduction pathway also plays a role in mediating the effects of TGF-β on eNOS transcription. We now show that TGF-β induction of eNOS is mediated in part by Smad2 interacting directly with a specific region of the eNOS promoter.
Materials and Methods
Recombinant human TGF-β was purchased from R&D Systems, Oxon, UK. The human eNOS promoter was a generous gift of Dr Thomas Michel (Brigham and Women’s Hospital, Boston, Mass).39 The Smurf cDNA was a kind gift from Dr X.-H. Feng (Baylor College of Medicine, Houston, Tex).
Bovine aortic endothelial cells (BAECs) and HUVECs were isolated using methods previously described.9 Human aortic endothelial cells (HAECs) were from Clonetics. Studies were performed on confluent monolayers at passages 2 to 7, made quiescent by serum deprivation with 0% calf serum for 16 hours.
To analyze the cellular localization of Smad2, we cultured BAECs, fixed cells with 4% (v/v) paraformaldehyde, permeabilized them with Triton X-100, incubated them with antibody to Smad2 for 1 hour, washed then 3 times with phosphate buffered saline, and incubated them with antibody to mouse IgG conjugated to FITC (Jackson Immunoresearch). Cells were then photographed with a fluorescent microscope.
Construction of Reporter Plasmids
The 1.6-kb fragment of the eNOS 5′-flanking region was a generous gift from Dr Thomas Michel (Brigham and Women’s Hospital, Boston, Mass). An eNOS promoter reporter plasmid was constructed by inserting the 1.6-kb fragment of the human eNOS promoter upstream of the firefly luciferase gene in the plasmid pGL3, as described previously.9 This promoter was designated −1624 eNOS-Luc. Deletion mutants of the eNOS promoter were generated by polymerase chain reaction (PCR), as described previously.9
eNOS Promoter Activity
Cell transfection was performed using methods modified from those previously reported.9 Photinus luciferase activity was determined using a luminometer and normalized against Renilla activity, using the Dual luciferase reporter kit (Promega), and normalized as relative luciferase light units/mg protein.
Electrophoretic Mobility Shift Assay
Proteins were prepared from nuclear extracts as described previously.54 Nuclear extracts from HAECs were incubated with radiolabeled eNOS promoter fragment at 22°C for 30 minutes. The mixture was then electrophoresed through a nondenaturing polyacrylamide gel. The gel was dried and autoradiographed.
Southwestern Blotting and Immunoblotting
Immunoblotting was performed as described previously.9 For Southwestern blotting, nuclear extracts (50 μg of total protein) from BAECs were electrophoresed through a SDS-10% polyacrylamide gel, transferred to a membrane, denatured with 6 mol/L guanidine, washed, and renatured with decreasing concentrations of guanidine HCl. The membrane was then blocked with 5% dry milk and incubated with [32P]end-labeled fragment of the eNOS promoter. The membrane was then washed and autoradiographed.
Data for promoter activity were analyzed by analysis of variance and Newman-Keuls post hoc testing. Results are expressed as mean±SEM, with a value of P<0.05 considered statistically significant.
TGF-β Stimulates eNOS Expression
To show that TGF-β increases eNOS expression in human endothelial cells, we treated HAECs with 2 ng/mL of TGF-β, and analyzed total RNA by Northern blotting with a radiolabeled eNOS cDNA probe. Densitometry was used to assess relative changes in signal intensity. TGF-β increases steady-state eNOS mRNA levels after 4 to 8 hours of treatment (Figure 1A). TGF-β increases steady-state eNOS mRNA levels both in proliferating endothelial cells by about 1.7-fold (Figure 1A, top) and confluent endothelial cells by about 3.6-fold (Figure 1A, bottom). Simultaneous treatment of HAECs with TGF-β and cyclohexamide (CHX) show that the effect of TGF-β on eNOS mRNA does not require new protein synthesis (Figure 1B).
TGF-β also increases steady-state levels of eNOS protein. Lysates of HUVECs treated with TGF-β were analyzed by immunoblotting with antibody to eNOS. TGF-β increases steady-state eNOS protein levels after 8 hours of treatment and produces a maximum level of eNOS protein 16 hours after treatment, with an increase over baseline of approximately 15-fold (Figure 1C).
TGF-β Transactivates the eNOS Promoter
We next explored the ability of TGF-β signaling to transactivate the eNOS promoter. Endothelial cells were transfected with a reporter vector, consisting of the 1624 bp of the human eNOS promoter upstream of the AUG start site, driving expression of the Photinus luciferase gene. (Cells were also transfected with a vector constitutively expressing Renilla luciferase, as an internal control for transfection efficiency.) Transfected cells were then treated with vehicle alone or TGF-β, and the amount of luciferase activity was measured. (Promoter transactivation is calculated as Photinus luciferase/Renilla luciferase/mg cell protein.) The eNOS promoter is activated at basal levels in both BAECs and HUVECs treated with vehicle alone (Figure 2A). TGF-β increases the transactivation of the eNOS promoter in BAECs and HUVECs (Figure 2A).
To determine the region of the eNOS promoter that mediates activation by TGF-β signaling, we next transfected BAECs with various reporter constructs containing 5′ deletions in the eNOS promoter upstream of Photinus luciferase. Transfected cells were then treated with TGF-β or vehicle, and the amount of luciferase activity was measured. TGF-β transactivates the −1500 to 0 bp region of the eNOS promoter (Figure 2B). TGF-β also transactivates the −1300 to 0 and the −1000 to 0 region of the eNOS promoter. However, TGF-β cannot transactivate the −720 to 0 region of the eNOS promoter. Thus, TGF-β induction of the eNOS promoter is mediated by the region of the eNOS promoter extending from −1000 to −720 bp.
TGF-β increases eNOS expression in part by inducing NF-1 to interact with an NF-1 element in the eNOS promoter.45 The human eNOS promoter contains two NF-1 sites, at −1219 to −1191 and at −947 to −935. Deletion of the region from −1300 to −1000 containing the −1219 to −1191 NF-1 site in the human eNOS promoter causes a slight decrease in the responsiveness to TGF-β, and deletion of the region from −1000 to −720 containing the −947 to −935 NF-1 site abolishes the response to TGF-β (Figure 2B). However, site-directed mutagenesis of the −947 to −935 NF-1 element of the eNOS promoter has a modest effect on TGF-β transactivation of the eNOS promoter (Figure 2C).
TGF-β Activates eNOS Promoter Binding Activity
To further explore the role of the −1000 to −720 bp region of the eNOS promoter in mediating TGF-β regulation of the eNOS promoter, we performed an electrophoretic mobility shift assay. HAECs were treated with media alone or TGF-β, nuclear extracts were isolated and incubated with radiolabeled eNOS promoter fragment extending from −1000 to −720, fractionated by SDS-PAGE, and autoradiographed. TGF-β activates a protein that interacts with the eNOS promoter fragment (Figure 3A).
To confirm that the −1000 to −720 bp fragment of the eNOS promoter mediates TGF-β transactivation of the eNOS promoter, we performed a Southwestern binding assay. BAECs were treated with TGF-β or vehicle alone, and nuclear extracts were prepared, fractionated by SDS-PAGE, and transferred to a membrane. Proteins on the membrane were renatured by sequential washes in buffer containing decreasing amounts of guanidine. The membrane was then incubated with a radiolabeled −1000 to −720 bp fragment of the eNOS promoter, washed, and autoradiographed. The eNOS promoter −1000 to −720 bp fragment binds to a polypeptide that appears in the nucleus 2 to 6 hours after TGF-β treatment (Figure 3B, left panel). This binding activity is competed by nonlabeled eNOS promoter fragment (Figure 3B, right panel). Thus TGF-β leads to the appearance in the nucleus of a polypeptide of approximately 55 kDa that interacts with a specific region of the eNOS promoter.
TGF-β Activates Smad2 in Endothelial Cells
Because Smad2 is a component of the TGF-β signaling cascade, we next tested the ability of TGF-β to activate Smad2 in endothelial cells. One hallmark of Smad2 activation is its translocation to the nucleus. We therefore treated BAECs with TGF-β, and then analyzed the localization of Smad2 in cells by immunofluorescence with an antibody to Smad2. Smad2 is located in the cytoplasm of resting BAECs, and TGF-β treatment causes transient translocation of Smad2 into the nucleus (Figure 4A). Smad2 then disappears from the nucleus 4 hours after TGF-β treatment (not shown). To confirm these results, we performed immunoblotting with antibody to Smad2 of cytosolic and nuclear extracts from BAECs treated with TGF-β. Smad2 is located in the cytosol but not the nucleus of resting BAECs, and Smad2 disappears from the cytosol and appears in the nucleus of cells within 2 hours of treatment with TGF-β (Figure 4B). TGF-β thus induces a translocation of Smad2 from the cytosol into the nucleus of endothelial cells.
Smad2 Stimulates Basal eNOS Expression and TGF-β-Stimulated Expression
We next explored the effect of Smad2 on eNOS expression. We first examined Smad2 expression in control and transfected cells. Smad2 is absent from the nucleus of resting cells, and TGF-β increases nuclear levels of Smad2 (Figure 5A, left). Transfection of BAECs with a FLAG-Smad2 expression vector increases Smad2 expression. FLAG-Smad2 is present in the nucleus of resting cells transfected with FLAG-Smad2 expression vector, and TGF-β increases nuclear levels of FLAG-Smad2 (Figure 5A, right).
Overexpression of Smad2 increases eNOS promoter transactivation. BAECs were cotransfected with empty vector or with vector-expressing FLAG-Smad2, and also with the eNOS promoter-luciferase reporter vector. Overexpression of Smad2 leads to transactivation of the eNOS promoter even in the absence of TGF-β (Figure 5B, vehicle-treated Control versus vehicle-treated Smad2). Furthermore, TGF-β increases the absolute level of transactivation of the eNOS promoter when Smad2 is overexpressed (Figure 5B, TGF-β-treated Control versus TGF-β-treated Smads). However, TGF-β treatment increases eNOS promoter activity over the activity in vehicle-treated cells, by approximately the same percentage in control or Smad2-overexpressing cells.
Finally, we measured the effect of Smad2 overexpression on endogenous eNOS expression. Steady-state protein levels of eNOS are higher in BAECs transfected with a Smad2 expression vector than in nontransfected cells (Figure 5C). TGF-β treatment increases eNOS protein levels even higher in BAECs overexpressing Smad2 than in control transfected BAECs (Figure 5C). Smad2 overexpression had no effect on steady-state levels of β-tubulin, demonstrating the specificity of TGF-β and Smad2 for regulation of eNOS expression (Figure 5C). These data all suggest that Smad2 increases eNOS expression.
Smad2 Interacts With eNOS Promoter
Because Smad2 transactivates the eNOS promoter and increases eNOS expression, we hypothesized that Smad2 interacts directly with the eNOS promoter. We performed an electrophoretic mobility shift assay to test this hypothesis. The addition of antibody to Smad2 blocks the ability of nuclear extracts to interact with the eNOS promoter fragment (Figure 3A). In addition, antibody to NF-1 affects the interaction of nuclear proteins with the eNOS promoter fragment, decreasing the intensity of a lower but not an upper band (Figure 3A).
A Southwestern binding assay confirmed our hypothesis that Smad2 interacts with the eNOS promoter region from −1000 to −720. Nuclear extracts from BAECs were fractionated by SDS-PAGE, transferred to a membrane, renatured, and then hybridized with a radiolabeled eNOS promoter −1000 to −720 bp fragment. Control BAECs do not contain binding activity for the eNOS promoter fragment (Figure 6, lane 1). TGF-β treatment of BAECs induces a polypeptide in the nucleus with eNOS promoter binding activity (Figure 6, lane 2). However, immunodepletion of these extracts with antibody to Smad2 abolishes this activity (Figure 6, lane 3). Finally, overexpression of Smad2 in TGF-β-treated BAECs increases this eNOS promoter binding activity (Figure 6, lane 4). Thus, Smad2 (or a complex containing Smad2) interacts directly with the eNOS promoter.
Smurf Interference With Smad2 Blocks TGF-β Activation of eNOS Expression
If Smad2 mediates TGF-β induction of eNOS expression, then interference with Smad2 should block the ability of TGF-β to activate the eNOS promoter. Smurf-2 modulates TGF-β signaling by activating the ubiquitination and destruction of Smad2.55 We therefore measured eNOS promoter activity in BAECs overexpressing Smurf-2. BAECs were cotransfected with the eNOS promoter reporter vector and with a vector overexpressing Smurf-2. Smad2 is present in nuclear extracts of control transfected BAECs (Figure 7A). Overexpression of Smurf-2 drastically reduces Smad2 steady-state protein levels, but overexpression of a mutant Smurf-2 has no effect on Smad2 levels (Figure 7A).
We then measured the effect of Smurf-2 on eNOS promoter activity. TGF-β increases eNOS promoter transactivation in control cells, as before (Figure 7B, left). However, TGF-β has no effect on the eNOS promoter when Smurf-2 is overexpressed (Figure 7B, middle). TGF-β is able to transactivate the eNOS promoter when a mutant Smurf-2 is expressed (Figure 7B, right). These data support the hypothesis that Smad2 mediates TGF-β induction of eNOS expression in endothelial cells.
The major finding of this study is that Smad2 mediates TGF-β regulation of eNOS expression. TGF-β activates Smad2 in endothelial cells, which translocates from cytoplasm to nucleus, where it interacts with the eNOS 5′-flanking region. Overexpression of Smad2 increases basal eNOS expression, and greatly increases TGF-β stimulation of eNOS expression. Finally, inhibition of Smad (by overexpression of Smurf) blocks TGF-β transactivation of the eNOS promoter.
TGF-β increases eNOS mRNA and protein levels within 4 to 8 hours of treatment (Figure 1). This rapid induction is consistent with the kinetics of phosphorylation and nuclear translocation of Smad2 after TGF-β binding to the signaling receptor complex (Figure 4). TGF-β induction of eNOS expression is independent of new protein synthesis (Figure 1B), which is also consistent with a signal transduction pathway involving a presynthesized messenger such as Smad2.
Our data show that Smad2 interacts with the eNOS promoter (Figures 3 and 6⇑). However, there is no Smad consensus binding element within the eNOS promoter. Although consensus Smad binding sites are absent from the eNOS promoter, the DNA binding specificity of Smads can be regulated by various partners that interact with Smads, such as Sp-1 and forkhead activin signal transducer-1.56–58⇓⇓ Despite the lack of a Smad binding element in the eNOS 5′-flanking region, Smad2 may be directed to specific regions of the eNOS promoter by binding partners.
Prior work showed that NF-1 mediates part of the effect of TGF-β on eNOS expression.45 Harrison and colleagues demonstrated that a region of the bovine eNOS promoter between −1269 to −935 is responsive to TGF-β. This prior study also showed that NF-1 binds to an NF-1 element from −1034 to −1006 within the bovine eNOS promoter region, and that NF-1 mediates TGF-β regulation of eNOS. The human eNOS promoter contains two potential NF-1 binding sites: one NF-1 element from −1219 to −1191 (corresponding to the bovine eNOS promoter −1034 to −1006), and a second NF-1 element from −947 to −935. Our data confirm the results of Harrison and colleagues45 that NF-1 plays a role in TGF-β regulation of eNOS. Our deletion mutation experiments show that the eNOS promoter region −1300 to −1000 containing the NF-1 site studied by Harrison and colleagues (bovine promoter −1034 to −1006, corresponding to human promoter −1219 to −1191) mediates some of the effects of TGF-β (Figure 2B). Our deletion experiments also show that the region containing the second NF-1 element, −1000 to −720, plays a more significant role in TGF-β regulation of eNOS (Figure 2B). Gel-shift experiments show that both NF-1 and Smad2 interact with this region of the eNOS promoter (Figure 3A). In addition, our point mutation experiments show that the second NF-1 site between −947 to −935 also mediates some of the effects of TGF-β (Figure 2C). Differences in the relative contributions of Smad2 and NF-1 to eNOS regulation in these two studies may be due in part to differences in bovine and human eNOS promoter sequences, and bovine and human endothelial cells. Thus, our data confirm the findings of Harrison and colleagues that NF-1 plays a role in TGF-β regulation of eNOS.
Identification of Smad2 as a regulator of eNOS expression suggests that pathways outside of the TGF-β signal transduction cascade can regulate eNOS as well. For example, shear stress activates Smad6, an inhibitory Smad, which can bind to Smad2 and prevent its transactivation of promoters.50 Shear stress can also activate MEKK-1, which in turn can activate Smad2 independent of TGF-β.59 Pathways that modulate the coactivator CREB binding protein (CBP) might also regulate eNOS transcription, because Smad2 interacts with CBP within endothelial cells.52,60⇓ Thus, Smad2 can serve as an integrator of multiple pathways that regulate eNOS expression.
Our findings might explain one aspect of the antiatherogenic properties of TGF-β. Recent evidence suggests that TGF-β inhibits atherogenesis in animals and humans.61,62⇓ These antiatherosclerotic effects of TGF-β may be mediated by multiple mechanisms. However, one possible pathway by which TGF-β reduces atherosclerosis is by activation of Smad2, followed by an increase in eNOS expression and NO synthesis. Increased NO, produced by higher levels of eNOS induced by Smad2, might in turn inhibit pathways leading to atherosclerosis.
This work was supported by grants from the Spanish Ministry (to M.S.), Spanish Government (to M.R.-P., DGES PM-97-0067; to D.R.-P., CICYT SAF-0054), and IRSIN (to D.R.-P. and M.R.-P.). This research was also supported by an American Heart Association Established Investigator Grant EIG 0140210N, National Institutes of Health grants P01 HL56091, P01 HL65608, NIH R01 HL53615, R01 HL63706, by the Ciccarone Center for the Prevention of Heart Disease, and by the Cora and John H. Davis Foundation (all to C.J.L.).
This manuscript was sent to Elizabeth G. Nabel, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received March 13, 2002; resubmission received September 6, 2002; revised resubmission received September 19, 2002; accepted September 20, 2002.
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text:
Publisher: ELEAH Enterprises
Date of Publication: March 30th 2016
Genre: Young Adult, Fantasy, Steampunk
Series: The Trinity Key Trilogy
Into Aether – Book 1
Escape Aether – Book 2
Save Aether – Book 3
Where you can find this book: Amazon
Her dad’s dead, her mom’s missing, and Theo is in danger.
Colorado girl Theodora (Theo) will do anything to find her missing mom, including travel into the hidden and mysterious Victorian subculture of Aether. She takes a ride with airship pirates to a floating island full of strange automatons and even stranger people.
After a century-old feud reignites, she uncovers the alarming truth about her family’s past. Finding her mother is more important than ever.
Ahhhhh…..steampunk!!!! Ahhhhh……Celtic mythology. Ahhhh…..a mix of both!!!
As you can see, I was pumped about reading a steampunk/Celtic mythology novel. Super duper pumped about reading this book and it didn’t let me down.
Theodora (or Theo as she liked to be called), was the perfect heroine and I loved her. She was the perfect combination of sass, sarcasm, angst, and innocence all rolled into one. She was the perfect definition of a teenager. I liked that she didn’t even blink when strange things started to happen to her. That whole cross-country flight/train ride/airship ride/floating island would have freaked me out. But not her and I admired that.
The steampunk part of the book didn’t happen until halfway through the book. I was in nerd heaven after that. Automans, half machine/half human people, odd way of talking, a freaking AIRSHIP!!!
The romance aspect was cute too. Theo was hilarious in fighting her feelings for Victor and when she gave in, well, I laughed. It was cute.
The whole Danu storyline was great too, along with the Order storyline. Lazarus was a creep and he got what he deserved.
The ending was a surprise to me. Not a cliffhanger but it did leave the book open for another book.
Oh, and since I never comment, I loved the cover!!! It was great!!
How many stars will I give Into Aether? 4
Why? This is a perfect combo of steampunk/romance and YA. Plus it had Celtic mythology and I love that!!!
Will I reread? Yes
Will I recommend to family and friends? Yes
Age Range: Young Adult
Why? No sex, minimal violence.
**I voluntarily reviewed a complimentary copy of this book**

domain: library


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PDP’s Rebuke of Tinubu’s Appeal
The Peoples Democratic Party (PDP) has strongly criticized President Bola Tinubu for his recent appeal against the release of his academic records to the PDP’s 2023 presidential candidate, Atiku Abubakar.
The party expressed its disapproval of Tinubu’s move in response to a legal suit filed by Atiku to obtain Tinubu’s academic records from Chicago State University (CSU).
Atiku’s Legal Pursuit
Atiku’s legal action was initiated to compel CSU to produce Tinubu’s academic records, as Tinubu had claimed to have attended the institution. Atiku argued that obtaining these records would strengthen his case challenging Tinubu’s victory in the election.
In response to Atiku’s request, US Magistrate Judge Jeffery Gilbert granted the order, compelling CSU to release Tinubu’s academic records.
Tinubu’s Appeal and Concerns
President Tinubu, however, filed an appeal against the order, citing potential harm to his interests if his academic records were released.
This move by Tinubu raised eyebrows and generated controversy surrounding the authenticity of his academic credentials.
In a subsequent development, Atiku filed another appeal in a US Court, seeking to overturn Tinubu’s objections to the release of his academic records.
This legal back-and-forth reflects the intensity of the dispute regarding the disclosure of Tinubu’s educational background.
PDP’s Reaction and Accusations
Reacting to these legal maneuvers, the PDP issued a statement through its spokesperson, Debo Ologunagba.
The party criticized Tinubu’s appeal, suggesting that it implies he has something to hide. The PDP questioned why Tinubu would oppose the release of academic records if he has consistently declared them to be impeccable.
Allegations Against APC and Supreme Court Fire Incident
The PDP’s statement did not stop at addressing Tinubu’s academic records but also criticized the All Progressives Congress (APC).
The PDP accused the APC of remaining silent in the wake of a fire outbreak at the Supreme Court, insinuating that the incident might be an attempt to frustrate electoral cases at the apex court.
The Controversy Continues
The legal battle over the release of President Tinubu’s academic records continues to be a focal point of contention in the political landscape.
The PDP’s condemnation of Tinubu’s appeal adds another layer of complexity to this ongoing dispute, with implications for the forthcoming elections and the public’s perception of political transparency and integrity.

domain: news


The artist's first song since last year's Boat Songs, one of the best albums of 2022.
July 18, 2023
The world is moving faster than ever before, but here are 30 songs that made time stand still for at least a few minutes.
June 14, 2023
2023 has felt like a transitional time for the music industry, but even in change, the last six months have provided some great albums.
June 12, 2023
Our March CoSign are set to take over indie with their latest LP.
April 7, 2023
With new album Rat Saw God, the Asheville band's days as indie's best-kept secret are over.
March 28, 2023
The zero-waste festival will also feature Madison Cunningham and Zambian psych rockers W.I.T.C.H.
March 22, 2023
Out April 7th, the Asheville band's follow-up to 2021's Twin Plagues also comes with a big North American and European tour.
January 18, 2023
The Asheville band just signed to Dead Oceans.
September 8, 2022
For fans of '90s indie rock icons like Pavement, My Bloody Valentine, or The Breeders.
August 13, 2021


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Iraq's health minister says between 100,000 and 150,000 civilians have been killed in the war, far more than other previously accepted figures.
Dozens of bodies are brought to morgues and hospitals daily
Officials say the total is based on estimates of the number of bodies brought to mortuaries and hospitals.
Casualty figures are a controversial topic, with estimates or counts ranging from 50,000 to 650,000 deaths.
No official count has ever been made public. The health ministry is run by supporters of a radical anti-US cleric.
Speaking during a visit to Vienna, Health Minister Ali al-Shamari said the figure was based on an estimate of 100 bodies being brought into government run mortuaries and hospitals every day.
In October, the UK medical journal The Lancet published a study saying nearly 655,000 Iraqis have died as a result of the war - a far higher death toll than other estimates.
The study was dismissed by President George W Bush and other US officials as not credible. It was based on cluster samples rather than body counts.
Previous counts, such as the Iraq Body Count, held that about 50,000 had people had been killed, based on partial figures from Iraqi institutions and media reports.
This figure was informally endorsed by senior American and Iraqi officials.
The US death toll stands at more than 2,800 troops
The head of the Baghdad central mortuary said on Thursday that he was receiving up to 60 victims of violent death - from insurgent violence and sectarian strife - each day at his facility alone.
Separately, the US military says three of its personnel have been killed in two separate incidents in Iraq.
Two soldiers were hit on Thursday by a roadside bomb in western Baghdad.
In Anbar province - a focal point of Sunni Arab resistance - the Americans say a marine died on Thursday of wounds sustained in fighting.
At least 23 US troops have been killed in November. In October, at least 105 soldiers were killed, the fourth highest monthly toll since US forces overthrew Saddam Hussein, and the worst for US casualties in nearly two years.


Tests on Microsoft Azure’s cloud have shown Mentor’s AMS simulation tools can run across thousands of processors with near-linear scaling.
Optima DA has launched a family of tools designed to speed up the analysis of radiation susceptibility in automotive SoC designs.
GlobalFoundries and TSMC have called off their legal battle with a wide-ranging patent cross-licensing deal.
The ESD Alliance is adding design and transportation-systems streams to the Semicon Europa 2019 show.
Different forms of heterogeneous integration take center stage at the IEEE International Electron Device Meeting (IEDM) in December this year.
October’s DVCon Europe conference will have an increased focus on the role of software in electronic systems and the challenges it poses to verification.
Arm is among a group of automotive OEMs and component suppliers who have set up a consortium to try to coordinate the development of autonomous-vehicle electronics.
Arm has relented on its opposition to custom instructions with the decision to let customers add them to V8-M processors.
UltraSoC has developed a bus monitor that will terminate transactions if it detects behavior that breaks rules set by a system designer.
Emulation is already playing a vital role in advanced automotive design within a digital twin environment.
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It’s the second day/night cricket match between New Zealand and England today at McLean Park, Napier, where a big crowd is expected.
It’s a series of three and the Black Caps one are already one up in the series after their close win in Hamilton last weekend.
They’ll be without form batsmen, Martin Guptill, due to a hamstring injury from the last match and also pace bowler, Mitchell McClenaghan, after sustaining an injury at the bowling crease in the last game where he left the field early after not completing his over.
Hamish Rutherford comes in for cover for Guptill and Tim Southee is making a return to replace McClenaghan.
Conditions for the match are looking good with the game set to start at 2 o’clock this afternoon.
Overhead and it should be mostly sunny this afternoon but cloudy areas could develop this evening but with no threat of rain.
Temperatures are set to be in the mid twenties mid-afternoon but cooling off to the 18-20 degree range tonight.
Winds are expected to northeasterly breezes or sea breezes which will help cool off both players and spectators a little.


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Can you even believe it is May already?? I can't. This year is just flying by. Since it is a new month, it's time for our sketch challenge at Die Cuttin' Divas. Here is this month's sketch:
Here is the card I made.
Paper: Echo Park "Anything Goes"
Elephant and balloon stamps: Simon Says Stamps
then cut out on my Scan N Cut.
Tag Die and sentiment: My Creative Time
Please stop by Die Cuttin Divas and check out what the rest of our talented design team members have made this week. We would love to have you join us this week. Remember to use a die cut on your project.
Thanks for stopping by my blog. Have a great week!!

domain: design


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Don't mind the girls. It's been proven that girls hate each other and their friendship is more brittle than an egg. Guys can fight and laugh it out the next day, girls can eye the same guy for one day and hate each other forever.
Girls have no friends from their own gender, and every male who talks to them wanna bone them. That explains their bitch mode and insecurity, not to mention that biologically, they need to live with their emotional shit fufilled, hence they are in a never ending vicious cycle of shit.

domain: sociology


ISLAMABAD: The federal government is to lift Covid-19 curbs from tomorrow (Tuesday) in cities with below 10 percent positivity ratio for at least three days.
According to the officials of the National Command and Operations Centre (NCOC), the restrictions were imposed following the start of the fifth wave of Covid-19 in January 2022 at a time when Covid-19 in major urban centres crossed 40 percent mark.
The curbs that are expected to be lifted include the resumption of regular classes and indoor dine-in as the infection rate has come down significantly nationwide, the officials said.
According to sources, “dine-in will be allowed with SOPs and children under 12 years would be allowed to start normal classes.”
Earlier, schools were allowed to open with 50 percent attendance (staggered days) for students below the age of 12 years. However, for students (fully vaccinated) over 12 years, the NCOC had recommended 100 percent attendance. Meanwhile, there was a complete ban on indoor dining. Meanwhile, Pakistan has witnessed a significant drop in the daily Covid-19 death count after over 10-day hike, the NCOC Covid-19-related data released here on Monday said.
According to the NCOC data, a total of 29 more deaths were recorded countrywide in the past 24 hours, taking the countrywide death count to 29,801. The NCOC data revealed that a total of 2,662 fresh Covid-19 cases were detected after performing 47,307 Covid-19 tests with a positivity ratio of 5.62 percent countrywide.
Copyright Business Recorder, 2022


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- Stamp Sets – Simply Succulents Bundle (156264)
- Card Stock – Soft Sea Foam (146988); Basic Black (121045); Basic White (159276); Granny apple Green (146990); Shimmery White (101910)
- Stamp Pads – Memento (132708)
- Stampin’ Blends:Flirty Flamingo (154884); Daffodil Delight (154883); Melon Mambo (153112); Balmy Blue (154830); Cinnamon Cider (153105); Mossy Meadow (154890)
- Misc. – Stampin’ Cut & Emboss (149653); Subtle 3D Embossing Folder (151775); Snail Mail Twine Combo Pack (154579); Dimensionals (104430); Multipurpose Liquid Glue (110755)
- Soft Sea Foam: 5 1/2″ x 8 1/2″, scored at 4 1/4″
- Basic Black: 4″ x 5 1/4″
- Whisper White: 3 7/8″ x 5 1/8″
- Granny Apple Green: 2 1/4″ x 5 1/8″
- Adhere the Basic Black and Whisper White layers to the card.
- Use the Subtle 3D Embossing Folder to add texture to the Oh So Ombre DSP, then adhere it to the card.
- Stamp the greeting onto a piece of Soft Sea Foam cardstock with Memento ink then cut out using the die.
- Stamp the flowers twice on a piece of Shimmery White cardstock. Color one of the flower bunches using Stampin’ Blends (you don’t have to color the large flower). Color just the large flower from the 2nd stamp images. Cut out the large flower using Paper Snips. Add the large flower to the flower bunch using dimensionals.
- Stamp the vase on a piece of Shimmery White cardstock using Memento ink. Color using Cinnamon Cider Stampin’ Blends.
- Wrap a piece of white twine around the vase 3 times and tie ends into a bow. (Tip: you can tape the twine on the back of the vase so that it stays in place.)
- Add vase and flowers to the card using Dimensionals.
- Add the greeting to the card using Multipurpose Liquid Glue.

domain: artistic


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Crain's 2012 Newsmaker of the Year
Thursday, February 21, 2013
On January 14, Crain's announced its Newsmaker of the Year for 2012 with a cover story. Past Newsmakers have included Mayor of Detroit, Dave Bing and Emergency financial manager of Detroit Public Schools, Robert Bobb; Congressman John Dingell, Richard Dauch, Edsel Ford II and Roger Penske.
The individual was honored at the Newsmaker of the Year Luncheon which was held on February 21 at Ford Field in Detroit.
Join the conversation on Twitter with #crainsnewsmaker
Date: Thursday, February 21, 2013
Time: 11:30 a.m. - 1:30 p.m.
Location: Ford Field, 1902 St Antoine St, Detroit, MI 48226 [Map]


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New album Honeymoon out September 18th. Pre-order now:
Official Store: http://lanadel.re/XWI6ZD
Sign up for updates: http://smarturl.it/LanaDelRey.News
More Lana Del Rey:
Elizabeth “Lizzy” Grant (born 21 June 1986), better known as Lana Del Rey, is a singer-songwriter from Lake Placid, New York, United States. Del Rey is known for embellishing her songs and music videos with an eerily nostalgic vibe heavy on Americana themes, including images of surfing, Coney Island, Frank Sinatra, and Marilyn Monroe. She has also listed a large number of her artistic influences, including Allen Ginsberg, Elvis Presley, Britney Spears, Nina Simone, Nirvana, Leonard Cohen, Bob Dylan… Read more about Lana Del Rey on Last.fm. User-contributed text is available under the Creative Commons By-SA License and may also be available under the GNU FDL.
female vocalists indie indie pop pop alternative
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Sam's grandfather vanished from his life the day Sam's father was buried. Now, ten years later, Grandpa Max wants to make amends. He sends letters that lead Sam on a scavenger hunt. Sam follows his grandfather's bizarre instructions though he's still not ready to forgive. To alleviate his anger at his grandfather, Sam turns to his favorite stress release: climbing onto roofs and leaving his tag, a spray painted symbol for Aquarius. When he gets caught by an elderly couple, Sam learns a valuable lesson about forgiveness. He's ready to forgive his grandfather, but is he too late?
"The book addresses weighty concepts succinctly, comprehensibly, and without pretense, and those looking for a hi-lo story with more depth will find it here."
"A wonderful novel that will draw reluctant readers in from the get-go and hold them captive until the bittersweet end. Denman's writing is clean and the voice brilliant. Highly Recommended."
"This book has a story that keeps the reader interested, with short chapters and easy-to-read vocabulary."
"Written for reluctant readers, this text tackles such heavy topics as forgiveness and death while using accessible, age-appropriate vocabulary."
"It's no small feat to engage and hold the interest of reluctant readers. With Rebel's Tag, Denman does the job."


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Minimalist Contemporary Home in Sentosa Cove, Singapore
The Bass Ensemble was completed in 2009 by the Singapore based studio Hyla Architects. This wonderful minimalist home is set over three stories, the roof and front façade have been clad in oxidized brass, the ground floor is entirely transparent with glass walls.
Bass Ensemble in Singapore, details by Hyla Architects:
“Our first project at Sentosa Cove, this bungalow presents a simple and subtle statement along Cove Way. Hidden behind a bamboo planting screen, the 1st storey is transparent – privacy being provided by a series of horizontal timber blinds.
These complement the oxidized brass roof that folds to become the front facade and also the canopy and carporch in one dynamic sweep. The living, dining and dry kitchen open across a covered timber terrace and an infinity lap pool. This maximises the entertainment possibilities at the 1st storey for our client.
The pool deck then cascades to a lower terrace level, before connecting to the seawall with it’s future yacht mooring on the water. A unique stair structure clad in timber connects the 3 levels.
The master bathroom utilises a barrel vaulted roof space and an oval skylight to create an interplay of light & shade. At the attic, the guest bathroom is seen as an extension of a luxe spa bath opening out onto a pebble garden on the roof terrace.”
Photos By: Derek Swalwell


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John Kasich has attacked Donald Trump relentlessly in debates and now his super PAC is planning to invest $2.5 million in the most aggressive takedown of the poll leader yet — on behalf of an increasingly anxious GOP establishment.
The attack, according to a blueprint shared with POLITICO, will play out over the next two months on radio, TV, mail and online in New Hampshire. Strategists with the pro-Kasich group, called New Day for America, say the budget for the anti-Trump campaign is likely to grow.
The offensive comes as some in the GOP are beginning to plot how to combat the real estate mogul and entertainer, who many are convinced would essentially deliver the White House to Democrats if he were the nominee. In launching the effort, the group hopes to position Kasich, who has lagged in the Republican contest and is searching for momentum, as a central Trump antagonist.
“We will be the tip of the spear against Trump,” said Matt David, a spokesman for the super PAC.
Rather than go after Trump for his business dealings or his past support for liberal causes, as some of his opponents have tried to do, the super PAC will depict Trump as someone who would be a deeply ineffective commander-in-chief and ill-suited for the demands of the Oval Office.
Fred Davis, the group’s colorful Hollywood-based ad-maker who is best-known for producing the “Demon Sheep” ad in the 2010 California Senate race, is working on a pair of anti-Trump TV ads. The commercials, David said, are designed to “accelerate what we believe would be buyers’ remorse” that would arise from a Trump presidency.
The group’s first volley came Thursday, when it released an ad that pictured the billionaire side by side with President Barack Obama. “On the job training for president does not work,” says the ad, which invokes last week’s tragic Paris terrorist attacks. The group is currently spending about $600,000 to air the commercial, though David said more airtime is being purchased.
For all the nervousness about Trump’s candidacy, however, few in the GOP have directed resources toward defeating him. Prominent Republican groups such as the Chamber of Commerce and the Karl Rove-founded American Crossroads, both establishment vehicles, have not spent any money against Trump. (In September and the beginning of October, the Club for Growth, an anti-tax group, spent $1 million to air a pair of TV commercials in Iowa that criticized the front-runner. The group has not done any since.)
Yet with the Iowa caucuses just a little more than two months away, and with Trump still riding high, the calculus may be changing. This week, as top party operatives and donors gathered in Las Vegas for the Republican Governors Association meeting, many contributors privately expressed unease about Trump’s consistent lead in early state and national polling.
Trump responded to the news by lashing out at Kasich on Twitter.
“I want to do negative ads on John Kasich, but he is so irrelevant to the race that I don’t want to waste my money,” Trump wrote in one tweet.
In launching the campaign, the pro-Kasich group says it is responding to money players who are eager to take Trump’s campaign down.
“Essentially, our donors are saying, ‘Enough is enough. Someone needs to take a stand,’” David said.
In the months leading up to the New Hampshire primary, which Kasich has made his focus, the group plans to air a mix of ads — some that attack Trump, some that contrast Trump with Kasich, and some that promote Kasich.
Kasich’s record as a two-term Ohio governor and as a House Budget Committee chairman, David argued, would make for a perfect contrast with Trump’s history of bombast. The two, he said, are “polar opposite.”
“Kasich isn’t going to make a promise he can’t keep,” David said.


text:
This article is only available in the PDF format. Download the PDF to view the article, as well as its associated figures and tables.
To the Editor:—
In the article, "Clinical Aspects of Eosinophilia in Atopic Disease" by Francis C. Lowell, MD (202:875, 1967) two points require discussion.The section Clinical Applications lists five general guides. Number three states that infection is probably absent in patients in asthmatic relapse who show eosinophilia, even when slight elevations of temperature and sedimentation rate occur with a purulent sputum containing large numbers of eosinophils. The fact that such patients may respond to full doses of steroids without antibiotics is beside the point. It has been known from the initial studies with steroids that inflammation in infection may be decreased by steroids; the fact that patients so treated "respond" is thus no evidence that infection is absent. On the contrary, I think most clinicians would agree that elevation of temperature, however slight, and the appearance of purulent sputum with or without eosinophils, should be considered strong clinical
Naterman HL. Eosinophilia In Atopic Disease. JAMA. 1968;203(11):991. doi:10.1001/jama.1968.03140110083030
* * SCHEDULED MAINTENANCE * *
The JAMA Network Sites will be conducting routine maintenance from 10/20/2017 through 10/21/2017. During this window access to content and authentication may be intermittently available. The JAMA Store will be completely unavailable during the maintenance window.

domain: literature


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Newswise — Charles Y. Liu, MD, PhD, a prominent expert in neurodegenerative diseases, can provide commentary on a new JAMA study showing an increased prevalence of chronic traumatic encephalopathy (CTE) diagnosed postmortem in former football players who donated their brains to science — including 110 of 111 National Football League athletes. CTE is a progressive neurodegenerative disease associated with repetitive head trauma.
Liu, professor of clinical neurological surgery at the Keck School of Medicine of USC and director of the USC Neurorestoration Center, can explain neuropathological features of CTE and the consequences of repetitive head trauma in football.
Liu is the principal neurosurgical consultant for the USC Trojans Athletics Department and a neurosurgical liaison for the Rose Bowl Game. He holds an MD from Yale University and a PhD in Chemical/Biomedical Engineering from Rice University. He trained in neurosurgery at the University of Southern California Affiliated Hospitals and completed a research visitorship in chemical engineering at the California Institute of Technology.
Liu’s principal research interests relate to restorative neurosurgery, including surgical, technological (advanced neuroprostheses, robotics) and biological strategies (stem cell transplantation) to restore function to the nervous system after injury due to trauma, stroke or neurodegenerative disease.
Liu has received numerous research awards, served many visitorships and delivered invited lectures around the world. He is editor-in-chief of the Journal of Clinical Neuroscience and serves on the editorial board of several neurosurgery and clinical neuroscience journals.

domain: medical


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Doctor Abbas Khan: Orthopaedic surgeon arrested on a humanitarian mission who died after a year in a Syrian prison
Belfast-born David McKittrick has been reporting on Northern Ireland since 1971, He has written for the East Antrim Times, the Irish Times and was The Independent's Irish correspondent for many years. He is the author of several books including Making Sense of the Troubles (2000) and Lost Lives (1999).
Friday 20 December 2013
Abbas Khan was a British doctor whose altruism took him to Syria to help with the long-running humanitarian crisis in that ravaged country. But he was plunged into a nightmare year of torture and confinement which ended in a lonely prison death.
Few accept the assertion of the Syrian government that he took his own life, particularly since he had been told he was only days away from being allowed home to Britain. The Syrian claim was utterly rejected by his family while the British government starkly declared that he was "in effect murdered." The Respect MP George Galloway, who had been negotiating with the Assad regime for his release, said it was inconceivable he had committed suicide, describing his death as "murder most foul."
The Khan family had been looking forward to welcoming him home for Christmas. According to his sister Sara he had said in a recent letter, "I can't wait to be back with you guys." They now describe themselves as "devastated and distraught." The family were also highly critical of the Foreign Office, complaining that they had received very little assistance and adding: "We are British citizens, and in our time of need the British government has forsaken us. My brother has been abandoned by his government."
The timing of the death raises important questions concerning the authority of President Assad, who had been personally involved in the decision to set the doctor free. The question arises: was someone in the government or intelligence apparatus intent on scuppering an Assad initiative towards a softer approach?
Abbas Khan was by no means the first medical casualty of Syria's civil war: the regime and various factions have been accused of targeting medical staff. Doctors and opposition organisations say more than 100 physicians have been killed while hundreds more have disappeared. In September the UN said it was alarmed by "the deliberate targeting of hospitals, medical personnel and transports."
Aged 32, from a family of Indian origin, Abbas Khan was born in London. He was married with two children, a six-year-old son, Abdullah, and a seven-year-old daughter, Rurayya. An orthopaedic surgeon, he worked at the Royal National Orthopaedic Hospital in Stanmore, north London and had previously worked at the Epsom and St Helier University Hospitals NHS Trust.
He left for the Middle East last year on a personal humanitarian mission after being moved by reports of huge numbers of civilian casualties. He flew out first to Turkey and then crossed into Syria, where his intention was to work as an emergency surgeon, but he was arrested within 48 hours of his arrival. It was some months before his family received confirmation that he was alive.
In letters home he said he had lost 40 to 50 per cent of his body weight due to repeated bouts of diarrhoea and chronic dermatological infection. When his mother Fatima was allowed to see him she found that "he was like a skeleton and could barely walk." He said he had been tortured and kept in dank cells infested with rats and lice. At one hearing, he said, he had been "accused of treating dying civilians, women and children – which has been classed as an act of terrorism."
Complaining of deteriorating mental health, he wrote that he had experienced "repeated and severe beatings, largely for no reason other than the pleasure of my captors." He suffered, he said, from "almost constant depression and suicidal ideation." He added: "I have been violently forced to beat other prisoners, kept in squalid conditions, denied access to toilets or medical treatment. I have also experienced male prisoners being beaten to death and female prisoners screaming as they were being abused."
His mother, who spent months in Damascus campaigning for his release, was told she could visit him on Monday last, but was then informed that he had died. The authorities insisted he had committed suicide by hanging himself with his pyjamas. His sister Sara said of him: "My brother is my hero. He didn't die in a normal way, he died trying to make a difference. Some could say he was naive for going out there and risking his life but he went out there because he knew his skills could help. He did something that he believed in and made an example for other people to do things."
His brother Afroze, who is also a doctor, said: "An innocent life was taken meaninglessly. He was the best brother I could have ever asked for and I know no one with a purer heart than him."
Abbas Khan, orthopaedic surgeon: born London 1981; married (two children); died Syria December 2013.
Apple agrees deal with Visa on contactless payments
- 1 Half of young women unable to ‘locate vagina’ and 65% find it difficult to say the word
- 2 Perez Hilton apologises for Jennifer Lawrence naked photo leak
- 3 A teacher speaks out: 'I'm effectively being forced out of a career that I wanted to love'
- 4 Mexican woman becomes world’s 'oldest person' at 127
- 5 Jennifer Lawrence 'naked sex video' will be leaked threatens 4Chan celebrity photo hacker
iPhone 6 'hidden code' could indicate sharper screens or bigger phones
Perez Hilton apologises for Jennifer Lawrence naked photo leak
Ariana Grande nude photos leak: 'These are completely fake' say singer's representatives
A teacher speaks out: 'I'm effectively being forced out of a career that I wanted to love'
Kate Upton nude pictures leak: Model's representatives 'looking into' authenticity of naked images
Rotherham child sex abuse scandal: Labour Home Office to be probed over what Tony Blair's government knew - and when
Robin Williams Emmys tribute led by Billy Crystal criticised for including 'racist' joke about Muslim woman
The Rotherham child abuse scandal is a tale of apologists, misogyny and double standards
What do immigrants really think of Britain? Polish immigrant's Reddit post goes viral
Do you realise just how foolish the UK looks?
With Douglas Carswell joining Ukip, my party has taken another giant step forward
£55000 - £60000 per annum + Benefits + Bonus: Harrington Starr: Lead C# Develo...
£40000 - £50000 per annum: Harrington Starr: You will not be expected to hav...
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The team at the CITF Secretariat has scoured numerous journals from around the world looking for answers about COVID, vaccines, and pregnancy. Here is what we’ve found:
People with COVID-19 who are pregnant are:
- 2.65 times more likely to require hospitalization and
- 5.46 times more likely to be admitted to ICU
than non-pregnant individuals with COVID-19.
Furthermore, COVID-19 infection has been associated with adverse pregnancy outcomes:
- The rate of preterm birth was 11.1% among those who had COVID-19 while pregnant, compared to only 6.8% of those who did not have the disease while pregnant;
- People with a laboratory-confirmed SARS-CoV-2 infection at the time they gave birth were more than twice as likely as uninfected people to have their baby be born prematurely or to die at birth.
Extensive research, including by CITF-affiliated researchers, has shown:
- COVID-19 vaccines are safe for pregnant people and their babies;
- Maternal vaccination offers benefits to the fetus, given that maternal antibodies are detected in the fetus within 16 days of vaccination;
- Infants born with maternal antibodies against COVID-19 could be protected in the first several months of their lives, when they are most vulnerable;
- At 6 months, 57% of infants born to mothers vaccinated with an mRNA vaccine had detectable antibodies compared with only 8% of infants born to unvaccinated mothers infected with COVID-19.
Antibodies can be transferred from mother to baby via the placenta and umbilical cord in utero, and through breast milk following birth. Research indicates that a mother being vaccinated in the late second or early third trimester may be optimal for high levels of anti-SARS-CoV-2 antibodies to be transferred to the baby before birth. Understanding the transfer of maternal antibodies to infants is important because maternal COVID-19 infections may account for a disproportionate burden of pediatric SARS-CoV-2–associated morbidity and vaccines are not currently available for infants younger than 6 months.
Pregnant people and their newborns are at increased risk of adverse events due to COVID-19
Pregnant people are at an increased risk of adverse effects from SARS-CoV-2 infection (the infection that causes COVID-19 disease) and are more likely to be admitted to an intensive care unit compared to non-pregnant individuals (1-4). Overall, people with COVID-19 who are pregnant are 2.65 times more likely to require hospitalization and 5.46 times more likely to be admitted to ICU than non-pregnant individuals with COVID-19 (4). COVID-19 has also been associated with increased risks of preeclampsia, preterm birth, and other adverse pregnancy outcomes (5). Thus, vaccination is an important tool for ensuring a healthy pregnancy.
Extensive research, including by CITF-affiliated scientists, has shown COVID-19 vaccines to be safe for pregnant people and their babies (6-11). Maternal vaccination also offers benefits to the fetus and the infant, a subject discussed below.
Increased risk of adverse events when the mother contracts SARS-CoV-2
A study from CANCOVIDPreg in Canada showed that SARS-CoV-2 infection during pregnancy was significantly associated with increased risk of adverse maternal outcomes and preterm births (4). The rate of preterm birth was 11.1% among COVID-19-affected pregnancies, compared to only 6.8% among those that were unaffected. This elevated risk was observed even for mild cases of COVID-19 (4). Similarly, in the UK, researchers found that prior to the widespread availability of vaccines, people with a laboratory-confirmed SARS-CoV-2 infection at the time they gave birth were more than twice as likely as those who were uninfected to have their baby die at birth or be born prematurely (12).
In a CITF-funded study from the Canadian National Vaccine Safety (CANVAS) Network (13), Drs. Julie Bettinger and Manish Sadarangani looked at health events requiring time off work or school or needing medical attention in vaccinated and unvaccinated pregnant people compared to vaccinated non-pregnant people. There was no significant association between vaccination status and health issues in those who were pregnant. They also highlighted that pregnant participants had fewer significant adverse events after vaccination than did similarly aged non-pregnant participants.
In a CITF-funded population-based cohort study in Ontario, researchers explored COVID-19 vaccination during pregnancy, compared with vaccination after pregnancy and with no vaccination. Vaccination was not significantly associated with increased risk of adverse peripartum outcomes (14).
Vaccination prior to and during pregnancy provides protective immunity to the fetus
Research has found that mRNA–based vaccines in pregnant people cause maternal antibody production as early as five days after the first dose. Transplacental transfer to the fetus was detected as early as 16 days after the first vaccination dose (15, 16). The increasing levels of maternal IgG and the transplacental transfer rate indicate that the timing between vaccination and birth may be an important factor to consider in vaccination strategies of pregnant people (15, 16).
A vaccine booster during the third trimester of pregnancy is also associated with strong maternal and neonatal humoral responses, as reflected by maternal and neonatal SARS-CoV-2 IgG antibody levels measured at birth (15). Overall, these findings support the administration of COVID-19 boosters to pregnant people to restore maternal and neonatal protective antibodies, given that they wane over time.
Maternal antibodies due to vaccination are transferred to the baby, offering the infant protection after birth
In addition to maternal protection against COVID-19, the mother’s vaccines provide various types of immunity to newborns, transferred via the placenta, umbilical cord, and breast milk. As Trostle emphasized in her 2021 article (16), if babies are born with antibodies against COVID-19, they could be protected in the first several months of their lives when they are most vulnerable.
A Morbidity and Mortality Weekly Report from February 2022 suggested completion of a two-dose mRNA series during pregnancy might help prevent COVID-19 hospitalization among infants aged <6 months (17).
Both spike- and RBD-specific IgG were detectable after maternal vaccination (18). Robust and comparable IgG titers were observed among vaccinated pregnant, lactating, and nonpregnant controls, all of which were significantly higher than those observed in pregnant people with previous SARS-CoV-2 infection. In their 2022 study published in JAMA, Shook and colleagues found that the majority of infants born to COVID-vaccinated mothers had persistent anti-S antibodies at six months compared with infants born to mothers who’d had a SARS-CoV-2 infection (19).
The findings below support the use of SARS-CoV-2 vaccines in pregnant people to protect both the mother and the child.
Immunity via the placenta and umbilical cord
Recent research suggests that maternal immunization provides neonatal protection through the transplacental transfer of antibodies (20). The newborn’s protection from infection is primarily dependent on neonatal innate immune responses and/or antibodies acquired via the mother’s placenta (21, 22). It is well established that IgG antibodies are actively transported to the fetus in the third trimester of pregnancy. A receptor on the placenta called Fcn is upregulated in the third trimester, thereby enabling antibodies present in the mother to be efficiently transferred to the fetus.
Among pregnant people vaccinated with Pfizer (BNT162b2), there was a demonstrated transfer of SARS-CoV-2 IgG across the placenta from the mother to their fetus with a positive correlation between maternal serum and cord blood antibody concentrations. In addition to maternal protection against COVID-19, the vaccine may also provide neonatal humoral immunity (22). At six months, 57% of infants born to mothers vaccinated with an mRNA vaccine had detectable antibodies compared with 8% of infants born to mothers with a COVID-19 infection (19).
Two doses of vaccine given to the mother resulted in significantly greater antibody persistence in infants than did a COVID infection in an unvaccinated mother (19). Two maternal SARS-CoV-2 vaccine doses during pregnancy led to significantly higher maternal and cord anti-SARS-CoV-2 IgG levels compared to one dose (18, 19, 23, 24). Antibody concentrations appear to be at least as high in infant cord blood as in the maternal circulation, and at least two vaccine doses during pregnancy are likely needed to reach maximal concentrations (25).
Emerging data demonstrate the presence of anti-SARS-CoV-2 IgG in umbilical cord blood following maternal vaccination with mRNA vaccines, with antibody transfer ratios (i.e. the ratio of maternal antibody to cord blood antibody) showing a strong correlation with both maternal antibody levels, time elapsed since vaccination, and whether one or both doses had been received (18, 24, 26, 27).
A booster shot was associated with augmented IgG levels in the blood, translating to the increased transfer of IgG to the fetus through the placenta (9).
Immunity via breast milk
Data suggest that maternal SARS-CoV-2-specific binding antibodies are efficiently transferred via breast milk following maternal mRNA vaccination, much like through the placenta (15, 17). Unlike in the placenta, however, where the antibody concentrations appear to be at least as high in infant cord blood as in the maternal serum, antibody concentrations are lower in breast milk (18).
Breast milk IgA rises rapidly following maternal vaccination, whereas IgG rises later, but may persist longer. Moreover, these antibodies retain a strong neutralizing capacity. At least two doses of vaccine appear to be required to reach maximal antibody concentrations in breast milk, as is the case for the cord blood (25). A booster shot was also associated with augmented IgG levels in the blood, translating to the increased transfer of IgG to the neonate through breast milk (9).
For protecting newborns, antibodies are efficiently transferred through breast milk, suggesting a potential prevention of infection in these newborns with undeveloped immune systems. There is clear evidence of SARS-CoV-2 specific IgA and IgG antibodies in breast milk for six weeks after vaccination. IgA secretion was evident as early as two weeks after vaccination, followed by a spike in IgG after four weeks (one week after the second vaccine). Antibodies found in breast milk showed strong neutralizing effects, suggesting a potential protective effect against infection in the infant (9, 15, 18, 25, 28-30).
Timing of maternal vaccination to give the baby the greatest immunity
Rottenstreich et al showed that vaccination of pregnant people early in the third trimester may enhance neonatal seroprotection (27). Because the transfer of maternal antibodies is most efficient in the third trimester, and it takes about three weeks for antibodies to peak after maternal vaccination, maternal SARS-CoV-2 immunization early in the third-trimester can enhance transplacental antibody transfer and increase neonatal neutralizing antibody levels (27).
Additionally, a study from Kugelman et al showed receipt of the Pfizer vaccine during the second trimester of pregnancy was also associated with maternal and neonatal humoral responses (31). Therefore, vaccination in the late second or early third trimester can lead to high levels of anti-SARS-CoV-2 antibodies being transferred to the baby before birth.
Further research on understanding the transfer of maternal antibodies to infants is important because COVID-19 infections may account for a disproportionate burden of pediatric SARS-CoV-2–associated morbidity and vaccines are not currently available for infants younger than 6 months (19).
- Lokken EM, Taylor GG, Huebner EM, Vanderhoeven J, Hendrickson S, Coler B, et al. Higher severe acute respiratory syndrome coronavirus 2 infection rate in pregnant patients. American Journal of Obstetrics & Gynecology. 2021;225(1):75.e1-.e16.
- Cassaniti I, Percivalle E, Zelini P, Ngaradoumbe Nanhorngue K, Parolo A, Bernardi V, et al. Both SARS-CoV-2 infection and vaccination in pregnancy elicited neutralizing antibodies in pregnant women and newborns. Clinical Microbiology and Infection. 2021;27(11):1708-9.
- Pirjani R, Hosseini R, Soori T, Rabiei M, Hosseini L, Abiri A, et al. Maternal and neonatal outcomes in COVID-19 infected pregnancies: a prospective cohort study. Journal of Travel Medicine. 2020;27(7).
- McClymont E, Albert AY, Alton GD, Boucoiran I, Castillo E, Fell DB, et al. Association of SARS-CoV-2 Infection During Pregnancy With Maternal and Perinatal Outcomes. Jama. 2022;327(20):1983-91.
- Wei SQ, Bilodeau-Bertrand M, Liu S, Auger N. The impact of COVID-19 on pregnancy outcomes: a systematic review and meta-analysis. Cmaj. 2021;193(16):E540-e8.
- De Rose DU, Salvatori G, Dotta A, Auriti C. SARS-CoV-2 Vaccines during Pregnancy and Breastfeeding: A Systematic Review of Maternal and Neonatal Outcomes. Viruses. 2022;14(3).
- Olson CK. COVID-19 vaccine safety in pregnancy: updates from the v-safe COVID-19 Vaccine Pregnancy Registry. 2021.
- Magnus MC, Gjessing HK, Eide HN, Wilcox AJ, Fell DB, Håberg SE. Covid-19 Vaccination during Pregnancy and First-Trimester Miscarriage. N Engl J Med. 2021;385(21):2008-10.
- Principi N, Esposito S. Is the Immunization of Pregnant Women against COVID-19 Justified? Vaccines. 2021;9(9):970.
- Zauche LH, Wallace B, Smoots AN, Olson CK, Oduyebo T, Kim SY, et al. Receipt of mRNA COVID-19 vaccines preconception and during pregnancy and risk of self-reported spontaneous abortions, CDC v-safe COVID-19 Vaccine Pregnancy Registry 2020-21. Res Sq. 2021.
- Fu W, Sivajohan B, McClymont E, Albert A, Elwood C, Ogilvie G, et al. Systematic review of the safety, immunogenicity, and effectiveness of COVID-19 vaccines in pregnant and lactating individuals and their infants. Int J Gynaecol Obstet. 2022;156(3):406-17.
- Gurol-Urganci I, Jardine JE, Carroll F, Draycott T, Dunn G, Fremeaux A, et al. Maternal and perinatal outcomes of pregnant women with SARS-CoV-2 infection at the time of birth in England: national cohort study. American Journal of Obstetrics & Gynecology. 2021;225(5):522.e1-.e11.
- Sadarangani M, Soe P, Shulha H, Valiquette L, Vanderkooi OG, Kellner JD, et al. Safety of COVID-19 vaccines in pregnancy: a Canadian National Vaccine Safety (CANVAS) Network study. medRxiv. 2022:2022.02.22.22271358 (Lancet Infectious Diseases, in press)
- Fell DB, Dhinsa T, Alton GD, Török E, Dimanlig-Cruz S, Regan AK, et al. Association of COVID-19 Vaccination in Pregnancy With Adverse Peripartum Outcomes. Jama. 2022;327(15):1478-87.
- Collier AY, McMahan K, Yu J, Tostanoski LH, Aguayo R, Ansel J, et al. Immunogenicity of COVID-19 mRNA Vaccines in Pregnant and Lactating Women. Jama. 2021;325(23):2370-80.
- Trostle ME, Aguero-Rosenfeld ME, Roman AS, Lighter JL. High antibody levels in cord blood from pregnant women vaccinated against COVID-19. American Journal of Obstetrics & Gynecology MFM. 2021;3(6).
- Halasa NB, Olson SM, Staat MA, Newhams MM, Price AM, Boom JA, et al. Effectiveness of Maternal Vaccination with mRNA COVID-19 Vaccine During Pregnancy Against COVID-19-Associated Hospitalization in Infants Aged <6 Months – 17 States, July 2021-January 2022. MMWR Morb Mortal Wkly Rep. 2022;71(7):264-70.
- Gray KJ, Bordt EA, Atyeo C, Deriso E, Akinwunmi B, Young N, et al. Coronavirus disease 2019 vaccine response in pregnant and lactating women: a cohort study. American Journal of Obstetrics and Gynecology. 2021;225(3):303.e1-.e17.
- Shook LL, Atyeo CG, Yonker LM, Fasano A, Gray KJ, Alter G, et al. Durability of Anti-Spike Antibodies in Infants After Maternal COVID-19 Vaccination or Natural Infection. JAMA. 2022;327(11):1087-9.
- Zdanowski W, Waśniewski T. Evaluation of SARS-CoV-2 Spike Protein Antibody Titers in Cord Blood after COVID-19 Vaccination during Pregnancy in Polish Healthcare Workers: Preliminary Results. Vaccines. 2021;9(6):675.
- Flannery DD, Gouma S, Dhudasia MB, Mukhopadhyay S, Pfeifer MR, Woodford EC, et al. Assessment of Maternal and Neonatal Cord Blood SARS-CoV-2 Antibodies and Placental Transfer Ratios. JAMA Pediatrics. 2021;175(6):594-600.
- Nir O, Schwartz A, Toussia-Cohen S, Leibovitch L, Strauss T, Asraf K, et al. Maternal-neonatal transfer of SARS-CoV-2 immunoglobulin G antibodies among parturient women treated with BNT162b2 messenger RNA vaccine during pregnancy. Am J Obstet Gynecol MFM. 2022;4(1):100492.
- Beharier O, Mayo RP, Raz T, Sacks KN, Schreiber L, Suissa-Cohen Y, et al. Efficient maternal to neonatal transfer of antibodies against SARS-CoV-2 and BNT162b2 mRNA COVID-19 vaccine. The Journal of clinical investigation. 2021;131(13).
- Mithal LB, Otero S, Shanes ED, Goldstein JA, Miller ES. Cord blood antibodies following maternal coronavirus disease 2019 vaccination during pregnancy. Am J Obstet Gynecol. 2021;225(2):192-4.
- Jorgensen SCJ, Burry L, Tabbara N. Role of maternal COVID-19 vaccination in providing immunological protection to the newborn. Pharmacotherapy. 2022;42(1):58-70.
- Prabhu M, Murphy EA, Sukhu AC, Yee J, Singh S, Eng D, et al. Antibody Response to Coronavirus Disease 2019 (COVID-19) Messenger RNA Vaccination in Pregnant Women and Transplacental Passage Into Cord Blood. Obstetrics & Gynecology. 2021;138(2).
- Rottenstreich A, Zarbiv G, Oiknine-Djian E, Vorontsov O, Zigron R, Kleinstern G, et al. Timing of SARS-CoV-2 vaccination during the third trimester of pregnancy and transplacental antibody transfer: a prospective cohort study. Clin Microbiol Infect. 2022;28(3):419-25.
- Perl SH, Uzan-Yulzari A, Klainer H, Asiskovich L, Youngster M, Rinott E, et al. SARS-CoV-2–Specific Antibodies in Breast Milk After COVID-19 Vaccination of Breastfeeding Women. JAMA. 2021;325(19):2013-4.
- Pace RM, Williams JE, Järvinen KM, Belfort MB, Pace CDW, Lackey KA, et al. Characterization of SARS-CoV-2 RNA, Antibodies, and Neutralizing Capacity in Milk Produced by Women with COVID-19. mBio. 2021;12(1).
- Romero Ramírez DS, Lara Pérez MM, Carretero Pérez M, Suárez Hernández MI, Martín Pulido S, Pera Villacampa L, et al. SARS-CoV-2 Antibodies in Breast Milk After Vaccination. Pediatrics. 2021;148(5).
- Kugelman N, Nahshon C, Shaked-Mishan P, Cohen N, Sher ML, Gruber M, et al. Maternal and Neonatal SARS-CoV-2 Immunoglobulin G Antibody Levels at Delivery After Receipt of the BNT162b2 Messenger RNA COVID-19 Vaccine During the Second Trimester of Pregnancy. JAMA Pediatr. 2022;176(3):290-5.


text:
Publications Search - Abstract View
||HPV16 seropositivity and subsequent HPV16 infection risk in a naturally infected population: comparison of serological assays.
||Lin SW, Ghosh A, Porras C, Markt SC, Rodriguez AC, Schiffman M, Wacholder S, Kemp TJ, Pinto LA, Gonzalez P, Wentzensen N, Esser MT, Matys K, Meuree A, Quint W, van Doorn LJ, Herrero R, Hildesheim A, Safaeian M, Costa Rican Vaccine Trial Group
||BB, CGB, MEB, IIB
||BACKGROUND: Several serological assays have been developed to detect antibodies elicited against infections with oncogenic human papillomavirus (HPV) type 16. The association between antibody levels measured by various assays and subsequent HPV infection risk may differ. We compared HPV16-specific antibody levels previously measured by a virus-like particle (VLP)-based direct enzyme-linked immunoassay (ELISA) with levels measured by additional assays and evaluated the protection against HPV16 infection conferred at different levels of the assays. METHODOLOGY/PRINCIPAL FINDINGS: Replicate enrollment serum aliquots from 388 unvaccinated women in the control arm of the Costa Rica HPV vaccine trial were measured for HPV16 seropositivity using three serological assays: a VLP-based direct ELISA; a VLP-based competitive Luminex immunoassay (cLIA); and a secreted alkaline phosphatase protein neutralization assay (SEAP-NA). We assessed the association of assay seropositivity and risk of subsequent HPV16 infection over four years of follow-up by calculating sampling-adjusted odds ratios (OR) and HPV16 seropositivity based on standard cutoff from the cLIA was significantly associated with protection from subsequent HPV16 infection (OR = 0.48, CI = 0.27-0.86, compared with seronegatives). Compared with seronegatives, the highest seropositive tertile antibody levels from the direct ELISA (OR = 0.53, CI = 0.28-0.90) as well as the SEAP-NA (OR = 0.20, CI = 0.06, 0.64) were also significantly associated with protection from HPV16 infection. CONCLUSIONS/SIGNIFICANCE: Enrollment HPV16 seropositivity by any of the three serological assays evaluated was associated with protection from subsequent infection, although cutoffs for immune protection were different. We defined the assays and seropositivity levels after natural infection that better measure and translate to protective immunity.


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text:
Pro-gun rights activists outbid cops for 'buy back' guns
'They're going to destroy firearms; we're going to put them in hands of citizens'
by Drew Zahn
While the Austin, Texas, police were offering grocery cards in exchange for unwanted firearms over the weekend, local activists showed up to outbid the men in uniform, insisting liberty would be better served if the guns were in the hands of law-abiding citizens instead.
At the "no-questions-asked" event held at Oak Meadow Baptist Church in South Austin, the Austin Police Department offered, for example, a $100 Visa grocery card for an unwanted handgun. The activists offered $110 in cash.
"We don't agree with the 'Guns for Groceries' program, because they're going to destroy most of the firearms," explained John Bush, executive director of the group Texans for Accountable Government. "The firearms that we purchase, we're going to put them in the hands of [Texans] who are in need of firearms to protect their families but they can't afford them."
Click here to read the entire article at WorldNetDaily.com.

domain: politics


text:
Microsoft announces Kinect for Windows, Newscorp apps for Xbox 360.
[newsimage]http://static.rankone.nl/images_posts/2012/01/LTsUtR.png[/newsimage]Consumer Electronics Show (CES) 2012 will be the last one where Microsoft will give a keynote, at least for now. The company didn't exactly go out with a bang at this CES but they did bring along some interesting announcements. The company used the giant electronics show to announce a slew of Newscorp apps for Xbox 360 as well as Kinect for Windows, which launches in February.
Read the full article here: [http://www.myce.com/news/microsoft-announces-newscorp-app-for-xbox-360-kinect-for-windows-57027/](http://www.myce.com/news/microsoft-announces-newscorp-app-for-xbox-360-kinect-for-windows-57027/)
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text:
By Ros Satar
- Serena Williams  v Svetlana Kuznetsova
- Timea Bacsinszky  v Garbiñe Muguruza
- Irina-Camelia Begu v Misaki Doi
- Madison Keys v Barbora Strycova
ROME, ITALY – The quarter-finals as World No. 1 Serena Williams leads the way into the last eight – We look at the pick of the four matches after the seeds scattered in the first three rounds.
Britwatch Tennis: Full Draws and Schedules
Serena Williams  v Svetlana Kuznetsova  – H2H: Williams leads 8-3
Williams looked like she might join the ranks of the seeds waiting for transport out of Rome after trailing an early break against compatriot and qualifier Christina McHale. Williams, back on court for the first time since Miami, had a comfortable enough straight sets win over Anna-Lena Friedsam in the opening round (benefiting from a bye in to the second round) but McHale was not shying away from the challenge.
From the start of the match she was putting Williams under pressure – forcing her to save three break points in the opening game before pressing home her advantage a few games later. Williams crucially broke back to level 5-5 before a see-saw tie-break saw set points evaporate and even drift to McHale’s side of the scoreboard, before edging back.
It was business as usual though for Williams who won six games in a row to seal her place in the last eight.
“I feel good, because I feel like she hits a lot of balls back,” she said in her post-match press conference, as reported by WTATennis.com. “I wanted to get a match in like that where she gets a lot of balls back and see how I was able to do against that and see how I was physically after that, too.
“I think physically, you know, every day is a new day for me, and I just try to see when I wake up. I just go from there.”
Kuznetsova fought through a tough three-setter against Aussie battler Daria Gavrilova in what had to be one of the best matches so far this tournament. The older Russian looked very sharp in the opening set, before Gavrilova produced some outstanding tennis to level things up.
It was a high quality tussle that saw Kuznetsova use all her experience to get past Gavrilova, and Williams knows this will be no easy match up between two ‘veterans’ of the tour.
She continued: “I think right now she’s probably one of the best clay-court players. I couldn’t ask for a better match going into Roland Garros, so this is a perfect opportunity for me, win or lose, to see where I am and get ready for what I’m ready for, which is the main event.”
Prediction: Williams in three sets
Timea Bacsinszky  v Garbiñe Muguruza
At the start of the clay court season, Muguruza had sounded confident of her return to the clay, calling it her ‘territory’ in Stuttgart, but has failed to stick together back to back wins on the dirt this year, until this week. She was merciless in her dispatch of Ekaterina Makarova, for the loss of just one game, and it looked as though she had the same in mind.
She breezed past Jelena Ostapenko in the first set, before the Latvian put up a little more of a fight in the second set, breaking the Spaniard for a 3-1 lead, but Muguruza kept her head down, eventually winning the last five games in a row to book her quarter-final berth.
Bacsinszky put paid to an all-Spanish quarter-final when she battled past Carla Suárez Navarro, coming from a set down and within two points of being on the wrong end of the score-line, eventually prevailing 5-7 7-5 6-2.
Bacsinszky is an all out battler, upending Simona Halep in fine style in Miami and of course booking her place here from behind. This will be a big test for Muguruza, as they gear up for some big hitting from two attacking players.
Prediction: Muguruza in three sets.
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domain: sports


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Belfast Royal Exchange bought by London-based developer
A major regeneration scheme in Belfast has been bought been a London-based developer.
Castlebrooke Investments has taken control of Royal Exchange, a swathe of properties between Royal Avenue and the Cathedral Quarter.
It was sold by the Cerberus investment fund which had control of the loans underlying those properties.
Castlebrooke said it is formulating new plans for a mixed use development.
The company said it would be working closely with Belfast City Council.
The council said it welcomed the news that the scheme had a new owner and hoped to see proposals for a revised project "come forward by early summer".
The original plan for Royal Exchange was drawn up the William Ewart group.
It included the construction of apartments, a hotel, offices and 50,000sq m of new retail floor space.
Its development costs were estimated at around £360m.
The scheme has planning permission but a development agreement with the Department for Social Development (DSD) lapsed last year.
The involvement of DSD is crucial because it has power to vest land and assemble all the property needed for the project.
The properties bought by Castlebrooke represent about 50% of the land needed for Royal Exchange as originally planned.
However, the scheme will be revised in light of other developments taking place in the city centre.
Royal Exchange has previously been proposed as a possible site for a John Lewis department store.
However, the retailer has only ever expressed an interest in opening a store at the Sprucefield retail park near Lisburn.