Improvement of phosphorus release from sludge by combined electrochemical-EDTA treatment

In this paper, combined with the addition of ethylenediaminetetraacetic acid (EDTA), the electrochemical treatment of waste activated sludge (WAS) was investigated to explore its effect on the release of phosphorus (P) from WAS. The results showed that during the electrochemical treatment, the addition of EDTA could significantly promote the release of P from the WAS to the supernatant, the optimal amount of EDTA was 0.4 g/g total suspended solids (TSS), when the release of total dissolved phosphorus (TDP), organic phosphorus (OP) and molybdate reactive phosphorus (PO43−-P) were 187.30, 173.84 and 13.46 mg/L, respectively. OP was the most likely form of P to be released during this process. Moreover, combined electrochemical-EDTA treatment could promote the release of P and metal ions from extracellular polymeric substances (EPSs) to the supernatant, and increase the solubility and disintegration of sludge. EDTA chelated the metal ions of sludge flocs and phosphate precipitates to cause sludge floc decomposition, thereby promoting the release of P from WAS.

Alternative method for trypsin-based cell dissociation using poly (amino ester) coating and pH 6.0 PBS

To maintain the cellular functions of a stem cells for therapeutic tissue engineering, an advanced cell culture method for safe cell dissociation is necessary. We developed a novel cell dissociation method by applying pH-responsive bioreducible polymer on the surface of tissue culture plates (TCPs). We applied acid-responsive bioreducible poly (amino ester) (PAE) as a new candidate for surface coating method to develop alternative cell dissociation method against conventional enzyme (trypsin-ethylene diamine tetra acetic acid (EDTA)) treatment. Human adipose derived stem cells (hADSCs) were cultured on and dissociated from PAE-coated TCPs to compare cell adhesion, cell proliferation, cell viability, and functionality to those of the cells cultured on and dissociated with trypsin-EDTA from normal TCPs without PAE coating. To confirm the in vivo therapeutic efficacy of the hADSCs retrieved from PAE-coated TCPs compared to that of the cells retrieved from normal TCPs with trypsin-EDTA, we induced skin defects at the dorsal area of mice and injected the cells collected from both conditions. With the PAE coating method, cell adhesion, cell proliferation, cell viability, and functionality, especially the angiogenic efficacy, were well preserved when compared to those of the cells treated with trypsin-EDTA. In addition to in vitro results, injecting hADSCs retrieved from PAE-coated TCPs showed similar in vivo angiogenesis and wound closing efficiency compared to those of injecting hADSCs retrieved from normal TCPs with trypsin-EDTA treatment at 2 weeks after the transplantation into mouse skin wound models. We proposed the alternative method for the cell dissociation with pH-responsive bioreducible polymer, PAE. This PAE coating method may lead to the development of alternative cell dissociation method without using enzyme for future regenerative medicine and stem cell therapy.

Disruption of the Metal Ion Environment by EDTA for Silk Formation Affects the Mechanical Properties of Silkworm Silk

Silk fiber has become a research focus because of its comprehensive mechanical properties. Metal ions can influence the conformational transition of silk fibroin. Current research is mainly focused on the role of a single ion, rather than the whole metal ion environment. Here, we report the effects of the overall metal ion environment on the secondary structure and mechanical properties of silk fibers after direct injection and feeding of silkworms with EDTA. The metal composition of the hemolymph, silk gland, and silk fiber changed significantly post EDTA treatment. Synchrotron FTIR analysis indicated that the secondary structure of silk fiber after EDTA treatment changed dramatically; particularly, the β-sheets decreased and the β-turns increased. Post EDTA treatment, the silk fiber had significantly decreased strength, Young’s modulus, and toughness as compared with the control groups, while the strain exhibited no obvious change. These changes can be attributed to the change in the metal ion environment in the silk fibroin and sericin in the silk gland. Our investigation provides a new theoretical basis for the natural silk spinning process, and our findings could help develop a method to modify the mechanical properties of silk fiber using metal ions.

Nanodiamond uptake in colon cancer cells: the influence of direction and trypsin-EDTA treatment

While some cell types readily ingest nanoparticles, others just don't. We report that, for certain cells, the uptake can be enhanced if the particles are administered from the basolateral side or if the cells are treated with trypsin-EDTA.

The Interaction of Cross-Linked Fibrin Fragment D-Dimer with αIIbβ3 Requires Receptor Activation, Occurs at Sites Other Than the γ-404-411 Sequence, and Contributes to Clot Retraction

Background: The interaction between the fibrinogen (fbg) γ-chain sequence 404-411 and the 'RGD pocket' formed by αIIb and β3 plays a major role in platelet (P) aggregation. It requires activation of αIIbβ3 when fbg is in solution, but not when fbg is immobilized. Less is known about the interaction of Ps with cross-linked fibrin, which presumably is the dominant form of fibrin(ogen) in human thrombi and the form that participates in clot retraction. αIIbβ3 is required for clot retraction since it is decreased or absent in Glanzmann thrombasthenia patients who lack a functional receptor. Paradoxically, clot retraction is essentially normal with fbg lacking γ-408-411 or in the presence EDTA, which eliminates fbg binding to the RGD pocket. We recently studied the interaction of αIIbβ3 with fbg fragments D100 (which has γ-404-411) and 'D98' (which essentially lacks γ-406-411) to identify fbg binding sites on αIIbβ3 other than the RGD pocket. We found that: 1. activated, but not unactivated, Ps adhere well to immobilized 'D98;' 2. cells expressing normal αIIbβ3 could bind to: a) fbg in the absence of EDTA, but not in the presence of EDTA, b) D100 in the absence or presence of EDTA, c) 'D98' in the presence, but not the absence, of EDTA; 3. cells expressing constitutively active αIIbβ3 mutants, but not cells expressing normal αIIbβ3, adhere well to immobilized 'D98.' These data support there being two separate mechanisms of binding, one involving the interaction of γ-404-411 (in fbg and D100) with the αIIbβ3 RGD binding pocket, and another involving a site on D100 and 'D98' (but cryptic in fbg) that interacts with a site on αIIbβ3 exposed by receptor activation or induced by EDTA. We have now studied adhesion of αIIbβ3 to the cross-linked fibrin fragment D-dimer (D-d). We predicted it to be similar to the interaction of 'D98' with αIIbβ3 because γ-K406, which is important in the binding of fbg to αIIb, participates in the antiparallel reciprocal factor XIIIa-mediated transglutaminase crosslinks between K406 and Q398 on two different γ-chains, and so is unlikely to be able to interact with the RGD pocket. Methods: D-d, purified from cross-linked fibrin after plasmin digestion, was obtained from Haematologic Technologies and found to contain a dominant band with the expected Mr and immunoblot pattern using antibodies to the individual fbg chains. Fibrinogen, 'D98,' and HEK293 cells expressing normal and mutated αIIbβ3 were prepared as we previously described (Zafar et al., Blood Advances 2017), and the adhesion assays were conducted as described in that paper. Clot retraction was performed by adding BSA, 'D98,' or D-d (200 µg/ml) to washed Ps (2 X 108/ml) + thrombin (0.2 U/ml) in a glass aggregometer cuvette. Results: αIIbβ3-mediated adhesion to immobilized D-d (10 µg/ml coating concentration) was similar to adhesion to 'D98' in that 1. Unactivated P adhered well to fbg, but poorly to 'D98' and D-d (n=4); 2. Activated Ps (10 µM thrombin receptor activating peptide) adhere ~2-fold better than unactivated Ps to fbg, but ~10-fold better to 'D98' and D-d (p<0.001 for unactivated vs activated Ps with both 'D98' and D-d; n=4); 3. HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) adhere well to fibrinogen, but not 'D98' and D-d; 4. HEK293 cells expressing the constitutively active aIIbF992A/F993Aβ3 receptor [αIIb(FF)β3-HEK] bound well to fbg, 'D98, and D-d (p<0.001 for both 'D98' and D-d compared to normal αIIbβ3; n=5); 5. EDTA treatment of Ps results in loss of adhesion to fbg, but significantly enhanced adhesion to 'D98' and D-d (p<0.05 for each ligand with and without EDTA; n=4); 6. EDTA treatment of αIIbβ3-HEK results in loss of adhesion to fbg, but significantly enhanced adhesion to both 'D98' and D-d (p<0.01 for each ligand with and without EDTA; n=5); 7. Adhesion of αIIb(FF)β3-HEK to D-d is like adhesion to 'D98' in being inhibitable by mAbs 7E3 and 10E5, eptifibatide, tirofiban, and RUC-4 (p<0.01 for all); n=4); 8. Adhesion of αIIb(FF)β3-HEK to D-d is similar to adhesion to 'D98' in being inhibitable by mutating β3 D119 to A in the MIDAS (p<0.01; n=6); 9. EDTA-induced adhesion of HEK-αIIbβ3 to both 'D98' and D-d is not inhibitable by mutating β3 D119 to A. 10. 'D98' and D-d inhibited clot retraction, but BSA did not. Conclusion: We conclude that cross-linked fibrin fragment D-dimer, like fibrinogen fragment 'D98', interacts with activated αIIbβ3 via sites other than the γ-404-411 peptide, and that this interaction contributes to clot retraction. Disclosures Coller: Rockefeller University: Patents & Royalties: RUC-4; Platelet Biogenesis: Consultancy; Centocor: Patents & Royalties: Abciximab; CeleCor: Consultancy, Equity Ownership; Accumetrics: Patents & Royalties: VerifyNow Assays; Scholar Rock: Consultancy, Equity Ownership.