scholarly journals Introducing Negatively Charged Residues on the Surface of Fetal Hemoglobin Improves Yields in Escherichia coli

2021 ◽  
Vol 9 ◽  
Author(s):  
Karin Kettisen ◽  
Leif Bülow
Keyword(s):  
Fetal Hemoglobin ◽  
Globin Chains ◽  
Γ Subunit ◽  
Alternative Protein ◽  
Free Heme ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
The Impact ◽  
Enhanced Yield ◽  
Culture Protocol

Fetal hemoglobin (HbF) has been developed into an important alternative protein for oxygen therapeutics. Such applications require extensive amounts of proteins, which only can be achieved via recombinant means. However, the expression of vertebrate hemoglobins in heterologous hosts is far from trivial. There are several issues that need to be dealt with. These include, among others, the solubility of the globin chains, equimolar expression of the globin chains, and access to high levels of free heme. In this study, we examined the impact of introducing negative charges on the surface of HbF. Three different HbF mutants were examined, carrying four additional negative charges on the α-subunit (rHbFα4), two additional negative charges on the γ-subunit (rHbFγ2) or a combination of these (rHbFα4/γ2). The increase in negative surface charge in these HbF mutants required the development of an alternate initial capture step in the downstream purification procedures. For the rHbFα4 mutant, we achieved a significantly enhanced yield of purified HbF with no apparent adverse effects on Hb functionality. However, the presence of non-functional Hb portions in the rHbFγ2 and rHbFα4/γ2 samples reduced the yields significantly for those mutants and indicated an imbalanced expression/association of globin chains. Furthermore, the autoxidation studies indicated that the rHbFγ2 and rHbFα4/γ2 mutants also were less oxidatively stable than rHbFα4 and wt rHbF. The study further verified the need for an improved flask culture protocol by optimizing cultivation parameters to enable yield-improving qualities of surface-located mutations.

scholarly journals Surface Exposure of PEG and Amines on Biodegradable Nanoparticles as a Strategy to Tune Their Interaction with Protein-Rich Biological Media

Nanomaterials ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 1354 ◽  
Author(s):  
Claudia Conte ◽  
Giovanni Dal Poggetto ◽  
Benjamin J. Swartzwelter ◽  
Diletta Esposito ◽  
Francesca Ungaro ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Electrostatic Interactions ◽  
Positive Charge ◽  
Particle Surface ◽  
Molecular Weights ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Body Compartments ◽  
Protein Rich ◽  
The Impact

Nanoparticles (NPs) based on amphiphilic block copolymers of polyethylene glycol (PEG) and biodegradable polyesters are of particular current interest in drug nanodelivery due to their easily manipulated properties. The interaction of these NPs with biological environments is highly influenced by shell features, which drive biological identity after administration. To widen the strategies available for tuning particle surface chemistry, here we developed a panel of amine-bearing PEGylated NPs with a poly(ε-caprolactone) (PCL) core for the delivery of lipophilic drugs, and investigated the impact of NP modifications on their interaction with abundant circulating proteins (human serum albumin—HSA—and mucin), as well as their transport through biological barriers (artificial mucus—AM, extracellular matrix—ECM). We prepared NPs based on a diamino-terminated PCL (amine-NPs) and its mixture with PEG-PCL copolymers (amine/PEG-NPs) at different PEG molecular weights by nanoprecipitation, as well as corresponding NPs of PEG-PCL (PEG-NPs). The presence of an amine-bearing polymer resulted in NPs with a net positive charge and a zeta potential dependent on the length of PEG in the copolymer. Amine/PEG-NPs had a larger fixed aqueous layer thickness as compared to PEG-NPs, suggesting that PEG conformation is affected by the presence of positive charges. In general, amine-bearing NPs promptly interacted with the dysopsonic protein HSA, due to electrostatic interactions, and lose stability, thereby undergoing time-related aggregation. On the other hand, amine/PEG-NPs interaction with mucin induced switching to a negative surface charge but did not alter the quality of the dispersion. The transport kinetics of NPs through a layer of artificial mucus and tumor extracellular matrix was studied by means of fluorescent NPs based upon FRET. Amine/PEG-NPs did not cross the ECM, but they were promptly transported through the AM, with swifter transport noted at increasing MWs of PEG in the copolymer. Finally, we demonstrated that all the different NP types developed in this study are internalized by human monocytes and, despite the positive charge, they did not induce a measurable inflammatory effect. In conclusion, we showed that the concurrent presence of both PEG and amine groups on NP surface is a promising strategy for directing their interaction with body compartments. While PEG-NPs are confirmed for their capacity to cross ECM-like compartments, amine/PEG-NPs are revealed as a powerful platform to widen the arsenal of nanotools available for overcoming mucus-covered epithelia.

Assessment of Damage to Human Platelets after Aggregation and Other Injuries by Microscopic Observation and Estimation of Serotonin Uptake

1970 ◽  
Vol 23 (02) ◽  
pp. 261-275 ◽  
Author(s):  
G Zbinden ◽  
J. N Mehrishi ◽  
S Tomlin
Keyword(s):  
Platelet Aggregation ◽  
Electrophoretic Mobility ◽  
Human Platelets ◽  
Freezing And Thawing ◽  
Low Degree ◽  
Microscopic Evaluation ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Charged Macromolecules ◽  
5 Ht Uptake

SummaryThe severity of platelet damage induced by hyper- and hypotonic NaCl solutions and freezing and thawing was assessed by microscopic evaluation and measuring inhibition of 5-HT uptake. The same techniques were used to quantitate the effects of aggregating agents. The positively charged macromolecules PS, Poly-L und Poly-O reduced the net negative surface charge as determined by microelectrophoresis, caused platelet aggregation and inhibited 5-HT uptake. The damaging effects of Poly-L and Poly-O were more severe and more closely related to concentration than that of PS. The negatively charged macromolecules Poly-IC and NaPS increased the anodic electrophoretic mobility. Poly-IC and heparin caused a low degree of platelet clumping and no inhibition of 5-HT uptake. NaPS produced severe platelet damage with extensive clumping and complete inhibition of 5-HT uptake. Na laurate had the same effect, but did not alter electrophoretic mobility. ADP caused concentration-dependent platelet aggregation and inhibition of 5-HT uptake. The effects of ADP and NaPS were compared in agitated and non-agitated platelet samples containing identical concentrations of the 2 compounds. Agitation was found to increase the degree of platelet clumping and to reduce 5-HT uptake.

Surface charge and extracellular polymer of sludge in the anaerobic degradation process

1996 ◽  
Vol 34 (5-6) ◽  
pp. 309-316 ◽  
Author(s):  
X. S. Jia ◽  
Herbert H. P. Fang ◽  
H. Furumai
Keyword(s):  
Surface Charge ◽  
Growth Phase ◽  
Anaerobic Degradation ◽  
Degradation Process ◽  
Anaerobic Sludge ◽  
Batch Experiments ◽  
High Substrate Concentration ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Extracellular Polymer

Changes of surface charge and extracellular polymer (ECP) content were investigated in batch experiments for three anaerobic sludges, each of which had been enriched at 35°C and pH 639-7.3 for more than 40 batches using propionate, butyrate and glucose, individually, as the sole substrate. Results showed that both ECP and the negative surface charge were dependent on the growth phase of microorganisms. They increased at the beginning of all batches when the microorganisms were in the prolific-growth phase, having high substrate concentration and food-to-microorganisms ratio. Both later gradually returned to their initial levels when the microorganisms were in the declined-growth phase, as the substrate became depleted. The negative surface charge increased linearly with the total-ECP content in all series with slopes of 0.0187, 0.0212 and 0.0157 meq/mg-total-ECP for sludge degrading propionate, butyrate and glucose, respectively. The change of surface charge for the first two sludges was mainly due to the increase of proteinaceous fraction of ECP; but, for glucose-degrading sludge, that could be due to the increases of both proteinaceous and carbohydrate fractions of ECP. The negative-charged nature of anaerobic sludge implies that cations should be able to promote granulation of anaerobic sludge.

scholarly journals Accuracy of Pulse Oximetry in the Presence of Fetal Hemoglobin—A Systematic Review

Children ◽  
2021 ◽  
Vol 8 (5) ◽  
pp. 361
Author(s):  
Ena Pritišanac ◽  
Berndt Urlesberger ◽  
Bernhard Schwaberger ◽  
Gerhard Pichler
Keyword(s):  
Pulse Oximetry ◽  
Neonatal Intensive Care ◽  
English Language ◽  
Arterial Oxygen Saturation ◽  
Fetal Hemoglobin ◽  
Arterial Oxygen ◽  
Cochrane Library ◽  
Preterm Neonates ◽  
Neonatal Blood ◽  
The Impact

Continuous monitoring of arterial oxygen saturation by pulse oximetry (SpO2) is the main method to guide respiratory and oxygen support in neonates during postnatal stabilization and after admission to neonatal intensive care unit. The accuracy of these devices is therefore crucial. The presence of fetal hemoglobin (HbF) in neonatal blood might affect SpO2 readings. We performed a systematic qualitative review to investigate the impact of HbF on SpO2 accuracy in neonates. PubMed/Medline, Embase, Cumulative Index to Nursing & Allied Health database (CINAHL) and Cochrane library databases were searched from inception to January 2021 for human studies in the English language, which compared arterial oxygen saturations (SaO2) from neonatal blood with SpO2 readings and included HbF measurements in their reports. Ten observational studies were included. Eight studies reported SpO2-SaO2 bias that ranged from −3.6%, standard deviation (SD) 2.3%, to +4.2% (SD 2.4). However, it remains unclear to what extent this depends on HbF. Five studies showed that an increase in HbF changes the relation of partial oxygen pressure (paO2) to SpO2, which is physiologically explained by the leftward shift in oxygen dissociation curve. It is important to be aware of this shift when treating a neonate, especially for the lower SpO2 limits in preterm neonates to avoid undetected hypoxia.

scholarly journals Farmers’ Perceptions of Commercial Insect-Based Feed for Sustainable Livestock Production in Kenya

2021 ◽  
Vol 13 (10) ◽  
pp. 5359
Author(s):  
Afrika Onguko Okello ◽  
Jonathan Makau Nzuma ◽  
David Jakinda Otieno ◽  
Michael Kidoido ◽  
Chrysantus Mbi Tanga
Keyword(s):  
Food Systems ◽  
Large Scale ◽  
Principal Component ◽  
Farm Income ◽  
Purchase Decisions ◽  
Wealth Status ◽  
Feed Formulation ◽  
Alternative Protein ◽  
Livestock Products ◽  
The Impact

The utilization of insect-based feeds (IBF) as an alternative protein source is increasingly gaining momentum worldwide owing to recent concerns over the impact of food systems on the environment. However, its large-scale adoption will depend on farmers’ acceptance of its key qualities. This study evaluates farmer’s perceptions of commercial IBF products and assesses the factors that would influence its adoption. It employs principal component analysis (PCA) to develop perception indices that are subsequently used in multiple regression analysis of survey data collected from a sample of 310 farmers. Over 90% of the farmers were ready and willing to use IBF. The PCA identified feed performance, social acceptability of the use of insects in feed formulation, feed versatility and marketability of livestock products reared on IBF as the key attributes that would inform farmers’ purchase decisions. Awareness of IBF attributes, group membership, off-farm income, wealth status and education significantly influenced farmers’ perceptions of IBF. Interventions such as experimental demonstrations that increase farmers’ technical knowledge on the productivity of livestock fed on IBF are crucial to reducing farmers’ uncertainties towards acceptability of IBF. Public partnerships with resource-endowed farmers and farmer groups are recommended to improve knowledge sharing on IBF.

scholarly journals Nanoemulsions for the Encapsulation of Hydrophobic Actives

Cosmetics ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 45
Author(s):  
Eduardo Guzmán ◽  
Laura Fernández-Peña ◽  
Lorenzo Rossi ◽  
Mathieu Bouvier ◽  
Francisco Ortega ◽  
...  
Keyword(s):  
Surface Charge ◽  
Long Term Stability ◽  
Promising Tool ◽  
Oil In Water ◽  
Nanometric Range ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Highly Hydrophobic ◽  
Technological Interest

This work analyzes the dispersion of two highly hydrophobic actives, (9Z)-N-(1,3-dihydroxyoctadecan-2-yl)octadec-9-enamide (ceramidelike molecule) and 2,6-diamino-4-(piperidin-1-yl)pyrimidine 1-oxide (minoxidil), using oil-in-water nanoemulsions with the aim of preparing stable and safe aqueous-based formulations that can be exploited for enhancing the penetration of active compounds through cosmetic substrates. Stable nanoemulsions with a droplet size in the nanometric range (around 200 nm) and a negative surface charge were prepared. It was possible to prepare formulations containing up to 2 w/w% of ceramide-like molecules and more than 10 w/w% of minoxidil incorporated within the oil droplets. This emulsions evidenced a good long-term stability, without any apparent modification for several weeks. Despite the fact that this work is limited to optimize the incorporation of the actives within the nanoemulsion-like formulations, it demonstrated that nanoemulsions should be considered as a very promising tool for enhancing the distribution and availability of hydrophobic molecules with technological interest.

scholarly journals RNA Delivery via DNA-Inspired Janus Base Nanotubes for Extracellular Matrix Penetration

MRS Advances ◽  
2020 ◽  
Vol 5 (16) ◽  
pp. 815-823
Author(s):  
Ian Sands ◽  
Jinhyung Lee ◽  
Wuxia Zhang ◽  
Yupeng Chen
Keyword(s):  
Extracellular Matrix ◽  
Small Rnas ◽  
Base Pairs ◽  
Major Barrier ◽  
Dna Base ◽  
Dna Base Pairs ◽  
Negative Surface ◽  
Negative Surface Charge ◽  
Delivery Vehicles ◽  
Low Cell Density

AbstractRNA delivery into deep tissues with dense extracellular matrix (ECM) has been challenging. For example, cartilage is a major barrier for RNA and drug delivery due to its avascular structure, low cell density and strong negative surface charge. Cartilage ECM is comprised of collagens, proteoglycans, and various other noncollagneous proteins with a spacing of 20nm. Conventional nanoparticles are usually spherical with a diameter larger than 50-60nm (after cargo loading). Therefore, they presented limited success for RNA delivery into cartilage. Here, we developed Janus base nanotubes (JBNTs, self-assembled nanotubes inspired from DNA base pairs) to assemble with small RNAs to form nano-rod delivery vehicles (termed as “Nanopieces”). Nanopieces have a diameter of ∼20nm (smallest delivery vehicles after cargo loading) and a length of ∼100nm. They present a novel breakthrough in ECM penetration due to the reduced size and adjustable characteristics to encourage ECM and intracellular penetration.

scholarly journals Hydroxyurea Treatment for Sickle Cell Disease

2002 ◽  
Vol 2 ◽  
pp. 1706-1728 ◽  
Author(s):  
Martin H. Steinberg
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Hemoglobin Concentration ◽  
Pharmacologic Therapy ◽  
Acute Chest Syndrome ◽  
Cell Disease ◽  
Globin Chains ◽  
Hydroxyurea Treatment ◽  
Erythroid Precursors

High fetal hemoglobin (HbF) levels inhibit the polymerization of sickle hemoglobin (HbS) and reduce the complications of sickle cell disease. Pharmacologic agents that can reverse the switch from γ- to β-chain synthesis — γ-globin chains characterize HbF, and sickle β-globin chains are present in HbS — or selectively increase the proportion of adult erythroid precursors that maintain the ability to produce HbF are therapeutically useful. Hydroxyurea promotes HbF production by perturbing the maturation of erythroid precursors. This treatment increases the total hemoglobin concentration, reduces the vaso-occlusive complications of pain and acute chest syndrome, and attenuates mortality in adults. It is a promising beginning for pharmacologic therapy of sickle cell disease. Still, its effects are inconsistent, trials in infants and children are ongoing, and its ultimate value — and peril — when started early in life are still unknown.

SURFACE-CHARGE-ENABLED PHOTOLYTIC HYDROGEN GENERATION IN V2O5·H2O/Au NANOCONJUGATES

MRS Advances ◽  
2016 ◽  
Vol 1 (46) ◽  
pp. 3121-3126
Author(s):  
Sunith Varghese ◽  
Charuksha Walgama ◽  
Mark Wilkins ◽  
Sadagopan Krishnan ◽  
Kaan Kalkan
Keyword(s):  
Surface Charge ◽  
Hydrogen Generation ◽  
Hydrogen Reduction ◽  
Energy Levels ◽  
Sol Gel ◽  
Band Edge ◽  
Conduction Band Edge ◽  
Current Voltage ◽  
Negative Surface ◽  
Negative Surface Charge

ABSTRACTThe present work investigates sol-gel synthesized vanadium oxyhydrate (V2O5·H2O) nanowires decorated with Au nanoparticles as potential photolytic H2 generators. As determined by UV photoelectron and optical spectroscopies, the conduction band edge of V2O5·H2O lies 0.6 eV below standard H+ reduction potential, implying no H2 can be generated. On the contrary, as measured by gas chromatography, our nanoconjugates yield reproducible light-to-hydrogen conversion efficiency of 5.3%, for the first hour of photolysis under 470 nm excitation. To explain the observed hydrogen reduction, we have hypothesized the vanadia electron energy levels are raised by some negative surface charge. With the objective of validating this hypothesis, we have performed cyclic current-voltage measurements. The derived conduction and valence band edge energies are not only consistent with the optical band gaps, but also validate the hypothesized energy increase by 1.6 eV, respectively. The negative surface charge is also corroborated by the ζ-potential. Based on the measured pH of 2.4, we attribute the negative surface charge to Lewis acid nature of the nanowires, establishing dative bonding with OH−. The present work establishes the importance of surface charge in photoelectrochemical reactions, where it can be instrumental and enabling in photolytic fuel production.

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