Publications by authors named "Shanny Ackerman"

6 Publications

  • Page 1 of 1

A Cell-Free Assay for Rapid Screening of Inhibitors of hACE2-Receptor-SARS-CoV-2-Spike Binding.

ACS Synth Biol 2022 04 4;11(4):1389-1396. Epub 2022 Apr 4.

We present a cell-free assay for rapid screening of candidate inhibitors of protein binding, focusing on inhibition of the interaction between the SARS-CoV-2 Spike receptor binding domain (RBD) and human angiotensin-converting enzyme 2 (hACE2). The assay has two components: fluorescent polystyrene particles covalently coated with RBD, termed virion-particles (v-particles), and fluorescently labeled hACE2 (hACE2F) that binds the v-particles. When incubated with an inhibitor, v-particle-hACE2F binding is diminished, resulting in a reduction in the fluorescent signal of bound hACE2F relative to the noninhibitor control, which can be measured via flow cytometry or fluorescence microscopy. We determine the amount of RBD needed for v-particle preparation, v-particle incubation time with hACE2F, hACE2F detection limit, and specificity of v-particle binding to hACE2F. We measure the dose response of the v-particles to known inhibitors. Finally, utilizing an RNA-binding protein tdPP7 incorporated into hACE2F, we demonstrate that RNA-hACE2F granules trap v-particles effectively, providing a basis for potential RNA-hACE2F therapeutics.
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http://dx.doi.org/10.1021/acssynbio.1c00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9003891PMC
April 2022

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection.

Mikrochim Acta 2021 02 4;188(3):67. Epub 2021 Feb 4.

Department of Biotechnology and Food Engineering, Technion - Israel Institute of Technology, Haifa, Israel.

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)-based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 μM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 μM vs. 2.7 μM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions.
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http://dx.doi.org/10.1007/s00604-021-04725-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862519PMC
February 2021

Progestin type affects the increase of heparanase level and procoagulant activity mediated by the estrogen receptor.

Hum Reprod 2021 01;36(1):61-69

Thrombosis and Hemostasis Unit, Rambam Health Care Campus, Haifa, Israel.

Study Question: Does progestin have an effect on heparanase level and procoagulant activity?

Summary Answer: Progestin increases the heparanase level and procoagulant activity via the estrogen receptor and the magnitude of the effect depends on the progestin type.

What Is Known Already: Users of combined oral contraceptives (COCs) containing third- and fourth-generation progestins have a higher risk of venous thrombosis compared to those employing second-generation progestins. Heparanase protein is capable of degrading heparan sulfate (HS) chains and enhancing activation of the coagulation system. We have previously demonstrated that estrogen enhances the expression and procoagulant activity of heparanase.

Study Design, Size, Duration: Estrogen and progestin receptor positive breast carcinoma cell lines: EMT6, T47D and MCF-7 were compared to the MDA-231 breast carcinoma cell line devoid of these receptors. This observational study incorporated 45 users of third-generation COCs progestins, 21 users of fourth-generation COCs progestins and 28 individuals not using hormonal therapy and not pregnant per history.

Participants/materials, Setting, Methods: Second-generation progestin-levonorgestrel, third-generation progestin-desogastrel (DSG), an estrogen receptor antagonist-ICI 182.780 and a progestin receptor antagonist-mifepristone, were added to cell lines. Heparanase level and procoagulant activity, HS levels, tissue factor (TF) activity and factor Xa levels were evaluated in the plasma of the study group.

Main Results And The Role Of Chance: Levonorgestrel and DSG increased heparanase levels in the cells and medium. The effect of DSG was more prominent and additive to that of estrogen. The effect was inhibited by ICI 182.780. In the plasma of COC users, heparanase procoagulant activity, HS levels, TF activity and factor Xa levels were significantly higher compared to controls. In COC pills containing the same dose of estrogen, the procoagulant effect of drospirenone was significantly stronger than that of DSG and gestodene.

Limitations, Reasons For Caution: The limitations of the study include a small number of participants in each study group, although the results are statistically significant and evaluated by several different coagulation parameters.

Wider Implications Of The Findings: The study demonstrates a new mechanism through which progestin affects coagulation system activation and shows that this effect is progestin type-dependent. Development of a progestin derivative with an attenuated effect on heparanase procoagulant activity may reduce thrombotic risk.

Study Funding/competing Interest(s): No external funding was sought for this study. Y.N. is named in a European patent application No. IL201200027 filed on 18 January 2012. Other authors have no conflict of interest to declare.

Trial Registration Number: N/A.
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http://dx.doi.org/10.1093/humrep/deaa263DOI Listing
January 2021

Heparanase Level and Procoagulant Activity Are Increased in Thalassemia and Attenuated by Janus Kinase 2 Inhibition.

Am J Pathol 2020 10 1;190(10):2146-2154. Epub 2020 Aug 1.

Thrombosis and Hemostasis Unit, Rambam Health Care Campus, Ruth and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel. Electronic address:

Patients with thalassemia exhibit an increased risk of thrombotic events that is augmented after splenectomy. Heparanase protein enhances cancer progression, angiogenesis, and inflammation; it also activates the coagulation system through direct interaction with tissue factor (TF). Additionally, erythropoietin, which is elevated in anemic patients, up-regulates heparanase expression via the Janus kinase 2 (JAK-2) pathway. This study aimed was to explore the heparanase profile in thalassemia. Coagulation factors were analyzed via immunostaining, enzyme-linked immunosorbent assay, and heparanase procoagulant activity assay. In spleen specimens of thalassemia major patients, a higher level of heparanase staining was observed compared with control spleens resected after trauma (P < 0.001). Higher heparanase levels, heparanase and TF procoagulant activity, and erythropoietin levels were found in the plasma of 67 thalassemia major patients compared with 29 control subjects. No difference was found in pediatric patients (23 of 67) compared with adults or splenectomized versus nonsplenectomized patients. Higher levels of heparanase, TF, TF pathway inhibitor, and TF pathway inhibitor-2 were observed in liver, spleen, heart, and kidney tissues of thalassemia intermedia mice (Hbb). These protein levels significantly reduced when mice were treated with the JAK-2 inhibitor ruxolitinib (P < 0.0001). In summary, heparanase levels are elevated in thalassemia, which may contribute to thrombotic phenomena in these patients. Inhibition of heparanase or the JAK-2 pathway may reduce thrombotic risk in thalassemia.
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http://dx.doi.org/10.1016/j.ajpath.2020.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520663PMC
October 2020

Circulating heparan sulfate chains and body weight contribute to anti-Xa levels in cancer patients using the prophylactic dose of enoxaparin.

J Thromb Thrombolysis 2020 Jul;50(1):112-122

Thrombosis and Hemostasis Unit, Rambam Health Care Campus, The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.

Hospitalized cancer patients are at increased risk of thrombosis and prophylaxis with heparin is recommended. Heparanase is a protein capable of degrading heparan sulfate (HS) chains. The first objective of the study was to examine the effects of weight on anti-Xa levels in cancer patients treated with a fixed dose of enoxaparin as thromboprophylaxis. The second aim was to assess a potential correlation between plasma pre-treatment coagulation parameters and anti-Xa levels in an assumption that heparanase degradation activity towards heparins and HS chains could affect anti-Xa levels. Two blood samples (prior to and 3 h after drug injection) of 76 cancer patients with an indication for prophylaxis with enoxaparin (40 mg) were evaluated for coagulation markers. Sub-prophylactic levels of anti-Xa (< 0.2 U/ml) were found in 16/76 (21%) patients; in 13/76 (13%) patients the values were supra-prophylactic (> 0.5 U/ml). In the subgroup of patients weighing > 80 kg, 7/14 (50%) individuals had a sub-prophylactic level. Overall, anti-Xa levels appeared to correlate with patient's weight (r = - 0.48, p < 0.0001), pre-treatment partial thromboplastin time (PTT), D-dimer, HS, heparanase levels and procoagulant activity. We concluded that plasma anti-Xa levels correlated with patient's weight. A substantial portion of cancer patients receiving enoxaparin prophylaxis was undertreated. For patients > 80 kg, a weight-adjusted prophylactic dose of enoxaparin could be considered. Elevated enoxaparin anti-Xa levels correlated with pre-treatment parameters of coagulation system activation. High pre-treatment HS and elevated plasma anti-Xa levels may potentially serve as biomarkers for the identification of patients at increased thrombosis risk.
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http://dx.doi.org/10.1007/s11239-020-02128-1DOI Listing
July 2020

Heparanase level and procoagulant activity are reduced in severe sepsis.

Eur J Haematol 2018 Feb 1;100(2):182-188. Epub 2017 Dec 1.

Thrombosis and Hemostasis Unit, Rambam Health Care Campus, The Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.

Background: During severe sepsis, levels and activity of all coagulation proteins are reduced. Heparanase is implicated in angiogenesis and tumor progression. We previously demonstrated that heparanase also affected the hemostatic system. It forms a complex and increases the activity of the blood coagulation initiator tissue factor.

Aim: To evaluate heparanase levels and procoagulant activity as predictors of sepsis severity.

Materials And Methods: Twenty-one patients with non-trauma, non-surgical sepsis admitted to the intensive care unit and 35 controls were recruited. Plasma samples were drawn from the study participants on days 1 and 7 following admission.

Results: Heparanase levels and procoagulant activity on day 1 were significantly reduced in patients compared to controls (P < .0001, P < .0001, respectively). Day 1 heparanase procoagulant activity ≥350 ng/mL yielded a negative predictive value for severe sepsis of 89%. Additionally, heparanase procoagulant activity on day 7 correlated with the change in the APACHE score between days 1 and 7 (r = .66, P = .007).

Conclusions: Heparanase procoagulant activity decreases during sepsis and returns to normal levels as soon as the patient recovers. Hence, it can be potentially used to predict the risk of severe sepsis. These findings need to be further explored in large-scale studies.
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http://dx.doi.org/10.1111/ejh.12997DOI Listing
February 2018
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