Abstract
We present the DNA-assisted control of enzymatic activity for the detection of a target protein using a new type of DNA–enzyme conjugate. The conjugate is composed of an enzyme inhibitor to regulate enzyme activity and a DNA aptamer to be responsive toward the analyte protein. Glutathione S-transferase (GST) and thrombin were selected as a model enzyme and an analyte protein. A hexahistidine tag was genetically attached to the C terminus of the GST, and the 5′ end of an oligonucleotide was conjugated with nitrilotriacetic acid (NTA) for the site-specific conjugation of the DNA with the GST based on a Ni2+ complex interaction. We found that fluorescein acted as a weak inhibitor of GST and succeeded in the regulation of GST activity by increasing the local concentration of the weak inhibitor by the hybridization of a 3′-end fluorescein-modified DNA. The catalytic activity of the DNA aptamer–enzyme conjugate showed a dose-dependent response to thrombin, indicating that the GST activity was clearly recovered by the binding of the DNA aptamer to thrombin. The current system enables the sensitive and specific detection of thrombin simply by measuring the enzymatic activity in a homogeneous medium.
Analytical Biochemistry
Volume 414, Issue 1, 1 July 2011, Pages 103-108
Monday
Researchers invent inkjet that prints out living skin
If you’ve ever seen the lesser-known Sam Raimi movie Darkman, you probably remember that the plot involved the main character, Dr. Westlake, trying to figure out a way to “print” liquid skin to help burn victims. Westlake never did figure out how to keep the synthetic skin from destabilizing past the 98 minute mark, but luckily, Wake Forest Instititute for Regenerative Medicine researchers seem to have mastered it, showing off their amazing skin printer that uses living cells instead of ink.
As the researcher note, “any loss of full-thickness skin of more than 4 cm in diameter will not heal by itself.” Enter their device, which allows a modified inkjet printer to produce reams of fresh skin which can be used to patch up victims of skin trauma. They’ve already tested it on mice, with extremely positive results.
How does the printer work? It has two heads: one dispenses skin cells mixed with a blood coagulent and type I collagen, and the other pumps out thrombin, which is another coagulate. Sprayed together, these chemicals create a reaction and form fibrin, which again helps to clot blood. On top of that, the printer then adds a layer of outer surface skin. Voila!
There’s still testing to be done — the next stage is on pigs, then human trials — but so far, this looks promising… if not to replace burned off skin, then at least to print out some life-like Halloween masks.
As the researcher note, “any loss of full-thickness skin of more than 4 cm in diameter will not heal by itself.” Enter their device, which allows a modified inkjet printer to produce reams of fresh skin which can be used to patch up victims of skin trauma. They’ve already tested it on mice, with extremely positive results.
How does the printer work? It has two heads: one dispenses skin cells mixed with a blood coagulent and type I collagen, and the other pumps out thrombin, which is another coagulate. Sprayed together, these chemicals create a reaction and form fibrin, which again helps to clot blood. On top of that, the printer then adds a layer of outer surface skin. Voila!
There’s still testing to be done — the next stage is on pigs, then human trials — but so far, this looks promising… if not to replace burned off skin, then at least to print out some life-like Halloween masks.
Wednesday
Von Willebrand factor and thrombin activation in children with newly diagnosed acute lymphoblastic leukemia: An impact of peripheral blasts†
Abstract
Background
The pathogenesis and the impact of therapy on thrombin activation in children with acute lymphoblastic leukemia (ALL) are unknown. Steroids may contribute to ALL-associated thrombosis. We explored the hemostatic effects of methylprednisolone monotherapy (MpMT) (32 mg/m2/day IV × 3 days) in children with newly diagnosed ALL.
Methods
Children (>1 to ≤18 years of age) enrolled on DFCI ALL05-01 protocol (n = 30; mean age 6.3 years), without prior steroid therapy, were eligible for study. Overnight fasting pre- and post-MpMT samples were analyzed for coagulation factors [FVIII:C, von Willebrand factor antigen (vWF:Ag) and fibrinogen] and parameters of thrombin generation [prothrombin fragments 1.2 (F1.2), thrombin–antithrombin complex (TAT), and D-dimer].
Results
At diagnosis F1.2 (1.5 nmol/L), TAT (10.9 µg/L), and D-dimers (2,766 ng/ml) levels were increased indicating endogenous thrombin activation. Patients with peripheral blasts (n = 17) had higher levels of vWF:Ag (1.89 vs. 1.14 P = 0.001), TAT (15.39 vs. 5.02 P = 0.038), and D-dimer (3,640 vs. 1,623 P = 0.019) compared to those without peripheral blasts. Following MpMT the blast count decreased significantly from 24% to 3.5% (P < 0.001) with reduction in level of vWF:Ag (1.5, P < 0.01), TAT (8.9, P = 0.42), and D-dimer (P = 0.018) despite 30% increase in FVIII:C levels (P = 0.005). However, patients without peripheral blasts had no significant change in vWF:Ag levels (1.14 vs. 1.25; P = 0.142) and had an increase in thrombin generation parameters.
Conclusions
We postulate that peripheral blasts through endothelial activation stimulate vWF:Ag production/secretion causing coagulation activation. Methylprednisolone therapy reduces the blast count and indirectly suppresses the coagulation activation. Future studies are required to confirm these findings. Pediatr Blood Cancer 2010;54:963–969
Uma Athale MD, MSc1,2,*, Albert Moghrabi MD3, Trishana Nayiager BScH, CCRP2, Yves-Line Delva RN3, Lehana Thabane PhD4,5,6, Anthony K.C. Chan MBBS1,2
Pediatric Blood & Cancer
Volume 54, Issue 7, pages 963–969, 1 July 2010
Background
The pathogenesis and the impact of therapy on thrombin activation in children with acute lymphoblastic leukemia (ALL) are unknown. Steroids may contribute to ALL-associated thrombosis. We explored the hemostatic effects of methylprednisolone monotherapy (MpMT) (32 mg/m2/day IV × 3 days) in children with newly diagnosed ALL.
Methods
Children (>1 to ≤18 years of age) enrolled on DFCI ALL05-01 protocol (n = 30; mean age 6.3 years), without prior steroid therapy, were eligible for study. Overnight fasting pre- and post-MpMT samples were analyzed for coagulation factors [FVIII:C, von Willebrand factor antigen (vWF:Ag) and fibrinogen] and parameters of thrombin generation [prothrombin fragments 1.2 (F1.2), thrombin–antithrombin complex (TAT), and D-dimer].
Results
At diagnosis F1.2 (1.5 nmol/L), TAT (10.9 µg/L), and D-dimers (2,766 ng/ml) levels were increased indicating endogenous thrombin activation. Patients with peripheral blasts (n = 17) had higher levels of vWF:Ag (1.89 vs. 1.14 P = 0.001), TAT (15.39 vs. 5.02 P = 0.038), and D-dimer (3,640 vs. 1,623 P = 0.019) compared to those without peripheral blasts. Following MpMT the blast count decreased significantly from 24% to 3.5% (P < 0.001) with reduction in level of vWF:Ag (1.5, P < 0.01), TAT (8.9, P = 0.42), and D-dimer (P = 0.018) despite 30% increase in FVIII:C levels (P = 0.005). However, patients without peripheral blasts had no significant change in vWF:Ag levels (1.14 vs. 1.25; P = 0.142) and had an increase in thrombin generation parameters.
Conclusions
We postulate that peripheral blasts through endothelial activation stimulate vWF:Ag production/secretion causing coagulation activation. Methylprednisolone therapy reduces the blast count and indirectly suppresses the coagulation activation. Future studies are required to confirm these findings. Pediatr Blood Cancer 2010;54:963–969
Uma Athale MD, MSc1,2,*, Albert Moghrabi MD3, Trishana Nayiager BScH, CCRP2, Yves-Line Delva RN3, Lehana Thabane PhD4,5,6, Anthony K.C. Chan MBBS1,2
Pediatric Blood & Cancer
Volume 54, Issue 7, pages 963–969, 1 July 2010
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