Human Transferrin Research Articles

A comparison of transferrin-receptor and epithelial cellular adhesion molecule targeting for microfluidic separation of cancer cells.

Microfluidic, flow cytometry, and immunomagnetic methods for cancer cell isolation have heavily relied on the Epithelial Cellular Adhesion Molecule (EpCAM) for affinity separation. While EpCAM has been used extensively for circulating tumor cell isolation, it cannot be used to isolate non-epithelial cells. The human transferrin receptor (CD71) can also be used for cancer cell isolation and has the advantage that as an affinity target it can separate virtually any cancer cell type, regardless of disease origin. However, direct comparison of the capture ability of EpCAM and CD71 has not been reported previously. In this work, cell capture with both EpCAM and CD71 were studied using a novel higher-throughput herringbone cell separation microfluidic device. Five separation chip models were designed and the one with the highest capture efficiency (average 90 ± 10%) was chosen to compare antigen targets for cell capture. Multiple cancer cell lines including CCRF-CEM, PC-3 and MDA-MB-231 were tested for cell capture performance using both ligands (anti-CD71 and anti-EpCAM) in the optimized chip design. PC-3 and MDA-MB-231 cells were spiked into blood at concentrations ranging from 0.5%-10%. PC-3 cells were separated by anti-CD71 and anti-EpCAM with 32-37% and 31-50% capture purity respectively, while MDA-MB-231 were separated with 35-53% and 33-56% capture purity using anti-CD71 and anti-EpCAM for all concentrations. The enrichment factor for the lowest concentrations of cells in blood ranged from 66-74X. The resulting enrichment of cancer cells shows that anti-CD71 was found to be statistically similar to anti-EpCAM for epithelial cancer cells, while anti-CD71 can be further used for non-epithelial cells, where anti-EpCAM cannot be used.

Exploring the molecular interactions of Galantamine with human Transferrin: In-silico and in vitro insight

The iron transporter protein, Transferrin (Tf), is a glycoprotein that binds and mediates the Iron (Fe) transport through blood plasma. It has a significant role in transporting Fe in the endosomal compartment of the cells by forming complexes of Fe-Tf-TfR (Transferrin receptor). The role of Tf in Alzheimer's disease (AD) pathogenesis is not so clear and needs to be explored, but it has been linked to the activity of Cyclooxygenase-2 (COX-2) in rats with diabetic neuropathy. Galantamine is a moderate inhibitor of Acetylcholinesterase (AChE) isolated from Galanthus woronowii. Here we have explored the possible interactions of Tf and Galantamine using computational as well as experimental approaches. First, we have performed molecular docking of Galantamine with Tf to predict their binding affinity and possible interactions. The molecular docking study was further escorted by all-atom molecular dynamics (MD) simulations for 100 ns followed by principal component analysis (PCA). MD studies suggested that the Tf-Galantamine docked complex is stable throughout the simulation trajectory. The computational results were further entrenched by a fluorescence-based binding study that suggested moderate binding of Galantamine with Tf with a binding constant (K) of 104 M−1. Isothermal titration calorimetry (ITC) further validated the spontaneous binding of Galantamine to Tf. This study provides a platform and can be implicated in AD therapy.

Transferrin receptor targeting by de novo sheet extension

The de novo design of polar protein-protein interactions is challenging because of the thermodynamic cost of stripping water away from the polar groups. Here, we describe a general approach for designing proteins which complement exposed polar backbone groups at the edge of beta sheets with geometrically matched beta strands. We used this approach to computationally design small proteins that bind to an exposed beta sheet on the human transferrin receptor (hTfR), which shuttles interacting proteins across the blood-brain barrier (BBB), opening up avenues for drug delivery into the brain. We describe a design which binds hTfR with a 20 nM Kd, is hyperstable, and crosses an in vitro microfluidic organ-on-a-chip model of the human BBB. Our design approach provides a general strategy for creating binders to protein targets with exposed surface beta edge strands.

Two peptides targeting endothelial receptors are internalized into murine brain endothelial cells.

The blood-brain barrier (BBB) is one of the main obstacles for therapies targeting brain diseases. Most macromolecules fail to pass the tight BBB, formed by brain endothelial cells interlinked by tight junctions. A wide range of small, lipid-soluble molecules can enter the brain parenchyma via diffusion, whereas macromolecules have to transcytose via vesicular transport. Vesicular transport can thus be utilized as a strategy to deliver brain therapies. By conjugating BBB targeting antibodies and peptides to therapeutic molecules or nanoparticles, it is possible to increase uptake into the brain. Previously, the synthetic peptide GYR and a peptide derived from melanotransferrin (MTfp) have been suggested as candidates for mediating transcytosis in brain endothelial cells (BECs). Here we study uptake, intracellular trafficking, and translocation of these two peptides in BECs. The peptides were synthesized, and binding studies to purified endocytic receptors were performed using surface plasmon resonance. Furthermore, the peptides were conjugated to a fluorophore allowing for live-cell imaging studies of their uptake into murine brain endothelial cells. Both peptides bound to low-density lipoprotein receptor-related protein 1 (LRP-1) and the human transferrin receptor, while lower affinity was observed against the murine transferrin receptor. The MTfp showed a higher binding affinity to all receptors when compared to the GYR peptide. The peptides were internalized by the bEnd.3 mouse endothelial cells within 30 min of incubation and frequently co-localized with endo-lysosomal vesicles. Moreover, our in vitro Transwell translocation experiments confirmed that GYR was able to cross the murine barrier and indicated the successful translocation of MTfp. Thus, despite binding to endocytic receptors with different affinities, both peptides are able to transcytose across the murine BECs.

Development and In Vitro Characterization of Transferrin-Decorated Nanoemulsion Utilizing Hydrophobic Ion Pairing for Targeted Cellular Uptake

PurposeThe aim of this study was to develop transferrin-conjugated nanoemulsions utilizing hydrophobic ion pairing for a targeted cellular uptake.MethodsIn the lipophilic phase of nanoemulsion composed of 60% oleic acid, 30% Capmul MCM EP and 10% Span 85, 1% cetyltrimethylammonium bromide (CTAB) and 3% phosphatidic acid (PA) were incorporated. After emulsification, the resulting droplets were decorated with human protein transferrin via hydrophobic ion pairing with PA and characterized regarding droplet size and zeta potential. Subsequently, cellular uptake of transferrin-conjugated nanoemulsion was investigated on Caco-2 and HeLa cell lines and determined by flow cytometry, cell lysis method and live cell imaging using confocal laser scanning microscopy.ResultsThe nanoemulsion showed a droplet size of 123.03 ± 2.1 nm and zeta potential of − 54.5 mV that changed because of the surface decoration with transferrin to 182.7 ± 0.2 and + 30.2 mV, respectively. Within the uptake studies utilizing flow cytometry, transferrin-conjugated nanoemulsion showed a 5.2-fold higher uptake in Caco-2 cells and twofold improvement in case of HeLa cells compared with unmodified formulation. The outcome was confirmed visually via live cell imaging.ConclusionAccording to the results, transferrin-conjugated nanoemulsion might be considered as a promising drug delivery system for a selective receptor-mediated drug delivery.

Naturally acquired blocking human monoclonal antibodies to Plasmodiumvivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolate and characterize anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies block PvRBP2b binding to reticulocytes and inhibit complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

Transferrin-based radiolabeled probe predicts the sensitivity of human renal cancer cell lines to ferroptosis inducer erastin

Ferroptosis induction has been recognized as a novel cancer therapeutic strategy. To effectively apply ferroptosis-targeting cancer therapy to individual patients, a diagnostic indicator for selecting this therapeutic strategy from a number of molecular targeting drugs is needed. However, to date, methods that can predict the efficacy of ferroptosis-targeting treatment have not been established yet. In this study, we focused on the iron metabolic pathway to develop a nuclear imaging technique for diagnosing the susceptibility of cancer cells to ferroptosis. As a nuclear probe, human transferrin (Tf) was labeled with Gallium-68 (68Ga) using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) as a chelator (68Ga-NOTA-Tf). Western blot assay and clonogenic survival assay with human renal cancer cell lines A498 and 786-O revealed that the protein expression level of transferrin receptor1 (TfR1) and sensitivity to a ferroptosis inducer, erastin, were correlated. A cellular uptake assay with 68Ga-NOTA-Tf revealed that the cancer cells sensitive to erastin highly internalized the 68Ga-NOTA-Tf. Furthermore, treatment with the TfR1 inhibitor ferristatin II reduced the cellular uptake of 68Ga-NOTA-Tf, indicating that the intracellular uptake of the probe was mediated by TfR1. These results suggest that 68Ga-NOTA-Tf can be useful in predicting the sensitivity of cancer cells to ferroptosis inducers.

Structural and molecular aspects of flavonoids as ligands for serum transferrin

Human serum transferrin (HST) acts as a carrier for Fe3+ and other ions. Binding of flavonoids to HST produces changes in the protein structure with direct implication on iron delivery into cells. We investigate the binding mechanism and affinity towards HST of three flavonoids: rutin, luteolin, and apigenin by different techniques: UV–Vis, fluorescence, fluorescence resonance energy transfer (FRET) combined with molecular docking. UV–Vis results indicate an interaction between flavonoids and HST. It was observed that HST fluorescence was quenched by these three flavonoids via a static process. All the interactions were moderate and the main driving forces are hydrophobic (ΔH > 0 and ΔS > 0) for rutin and luteolin binding or electrostatic (ΔH < 0 and ΔS > 0) for apigenin binding. FRET and molecular docking studies confirm the fluorescence static quenching mechanism by flavonoid binding. The binding of all three flavonoids increases HST stability. These results present the potential use of HST in target-oriented delivery of flavonoids and possibly other drugs into cells.

Chemically-Tuned Solid-State Nanopores for Single-Molecule Biophyics

Solid-state nanopores (SSNs) are nanofluidic conduits fabricated through synthetic materials such as silicon nitride, silicon dioxide, graphene, etc. Intrapore variations, temporal fluctuations in open-pore current and analyte sticking are legacy limitations associated with SSNs which have challenged its footprint in single molecule science. We report a modification to the Controlled Dielectric Breakdown (CDB) method of nanopore fabrication on silicon nitride (SiNx) membranes where a blend of electrolyte and sodium hypochlorite is used as the conducting solution. These chemically tuned SSNs are devoid of the fundamental challenges associated with SSNs. The change in surface chemistry, with ∼3 orders of magnitude weaker Ka compared to conventional CDB nanopores, is thought to be inextricably linked with the Ohmic nature, extremely stable open-pore current during analyte translocation (hours), minimum analyte sticking with self-correction of current signal (if analyte sticking causes current deviation) and higher analyte responsiveness. The transport phenomena through these chemically modified nanopores are successfully tested by translocating 1 kb dsDNA and the human serum transferrin (hSTf) protein, with the highlight being the yield of an excess of 200 thousand DNA translocation events. Moreover, a comprehensive noise analysis done for the low frequency regime of this new class of SSNs revealed interesting characteristics which are distinct from the conventionally fabricated SSNs.

Application of Localized Surface Plasmon Resonance Spectroscopy to Investigate a Nano-Bio Interface.

The accurate determination of events at the interface between a biological system and nanomaterials is necessary for efficacy and safety evaluation of novel nano-enabled medical products. Investigating the interaction of proteins with nanoparticles (NPs) and the formation of protein corona on nanosurfaces is particularly challenging from the methodological point of view due to the multiparametric complexity of such interactions. This study demonstrated the application of localized surface plasmon resonance (LSPR) spectroscopy as a low-cost and rapid biosensing technique that can be used in parallel with other sophisticated methods to monitor nano-bio interplay. Interaction of citrate-coated gold NPs (AuNPs) with human plasma proteins was selected as a case study to evaluate the applicability and value of scientific data acquired by LSPR as compared to fluorescence spectroscopy, which is one of the most used techniques to study NP interaction with biomolecules. LSPR results obtained for interaction of AuNPs with bovine serum albumin, glycosylated human transferrin, and non-glycosylated recombinant human transferrin correlated nicely with the adsorption constants obtained by fluorescence spectroscopy. This ability, complemented by its fast operation and reliability, makes the LSPR methodology an attractive option for the investigation of a nano-bio interface.

Analysis of protein denaturation, aggregation and post-translational modification by agarose native gel electrophoresis

Agarose native gel electrophoresis has been developed to separate proteins and protein complexes in the native state. Here, we applied this technology to analyze proteins that undergo degradation, post-translational modification or chemical/physical changes. Antibodies showed aggregation/association upon acid or heat treatment. Limited reduction of disulfide bonds resulted in non-covalent aggregation of bovine serum albumin and cleavage of only inter-chain linkages of an antibody that had no effects on its overall structure. Native agarose gel analysis showed changes in mobility of human transferrin upon Fe3+ binding. Analysis of a commercial glycated human hemoglobin A1c showed no difference in electrophoretic pattern from un-modified hemoglobin. Native agarose gel showed aggregation of a virus upon acid or heat treatment. We have extracted bands of bovine serum albumin from the agarose native gel for sodium dodecylsulfate gel electrophoresis analysis, showing degradation of aged sample. Lastly, we analyzed phosphorylation of Zap70 kinase by native gel and Western blotting. These applications should expand the utility of this native gel electrophoresis technology.

Magnetospirillum magneticum as a Living Iron Chelator Induces TfR1 Upregulation and Decreases Cell Viability in Cancer Cells.

Interest has grown in harnessing biological agents for cancer treatment as dynamic vectors with enhanced tumor targeting. While bacterial traits such as proliferation in tumors, modulation of an immune response, and local secretion of toxins have been well studied, less is known about bacteria as competitors for nutrients. Here, we investigated the use of a bacterial strain as a living iron chelator, competing for this nutrient vital to tumor growth and progression. We established an in vitro co-culture system consisting of the magnetotactic strain Magnetospirillum magneticum AMB-1 incubated under hypoxic conditions with human melanoma cells. Siderophore production by 108 AMB-1/mL in human transferrin (Tf)-supplemented media was quantified and found to be equivalent to a concentration of 3.78 µM ± 0.117 µM deferoxamine (DFO), a potent drug used in iron chelation therapy. Our experiments revealed an increased expression of transferrin receptor 1 (TfR1) and a significant decrease of cancer cell viability, indicating the bacteria’s ability to alter iron homeostasis in human melanoma cells. Our results show the potential of a bacterial strain acting as a self-replicating iron-chelating agent, which could serve as an additional mechanism reinforcing current bacterial cancer therapies.

The role of transferrins and iron-related proteins in brain iron transport: applications to neurological diseases.

Iron transport in the central nervous system (CNS) is a highly regulated process in which several important proteins participate to ensure this important metal reaches its sites of action. However, iron accumulation has been shown to be a common factor in different neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Multiple Sclerosis, and Sanfilippo syndrome. This review is divided into four parts. The first part describes brain iron transport in homeostasis, mentioning the main proteins involved, whereas the second part contrasts the consequences of iron dysregulation, elaborating on its role in the aforementioned neurodegenerative diseases. The third part details the functions of the main proteins involved in brain iron homeostasis and their role in neurodegeneration. In the fourth part, in order to highlight the importance of transport proteins, the focus is set on human serum transferrin, the main iron transport protein. This final part describes perspectives about the mechanisms and chemical properties of human transferrin for the development of potential targeted drug delivery systems across the blood-brain barrier (BBB) or enhancers for the treatment of neurological diseases.

Luminescent naphthalimide-tagged ruthenium(ii)-arene complexes: cellular imaging, photocytotoxicity and transferrin binding.

Two water-soluble piano-stool shaped ruthenium(ii)-arene complexes, [RuII(η6-p-cymene)(L)Cl2] [RuLCl] and [RuII(η6-p-cymene)(L)(PTA)Cl] [RuLPTA], were designed as emissive photocytotoxic agents tagged with morpholine as the lysosome targeting moiety. Here, L = N-(2-morpholinoethyl)-4-(2-aminoethyl)amino-naphthalimide, and PTA = 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decane. The crystal structure of [RuLCl] exhibits the pseudooctahedral 'three-legged piano-stool' geometry, wherein Ru(ii) is bound to the η6-p-cymene moiety as a base and two chlorides and the amine-N of the ligand L occupies the three legs of the stool. The complexes exhibited both the possibility of covalent adduct formation via the hydrolyzed Ru-Cl bond and non-covalent intercalation binding through planar naphthalimide moieties. The complexes showed enhanced photo-cytotoxicity under low-power blue LED light irradiation (λmax = 448 nm) mediated by 1O2, thereby acting as potential PDT agents. Fluorescence microscopy studies revealed that luminescent complexes preferentially localized in both the lysosomes and nucleus for effectively targeting and damaging the nuclear DNA for PDT effects. Due to enhanced lipophilicity of [RuLCl], it showed higher internalization into MCF-7 cell, measured in terms of the ruthenium content using ICP-MS. The interaction of the complexes with human transferrin (hTf) proteins was studied through molecular docking calculations, suggesting favorable binding through histidine residues and possible internalization into cancer cells via TfR-mediated endocytosis. The luminescence properties of the complexes were well-utilized to study their cellular uptake mechanism via endocytosis using fluorescence microscopy.

Polymer Brush-Grafted Nanoparticles Preferentially Interact with Opsonins and Albumin.

Nanoparticles find increasing applications in life science and biomedicine. The fate of nanoparticles in a biological system is determined by their protein corona, as remodeling of their surface properties through protein adsorption triggers specific recognition such as cell uptake and immune system clearance and nonspecific processes such as aggregation and precipitation. The corona is a result of nanoparticle–protein and protein–protein interactions and is influenced by particle design. The state-of-the-art design of biomedical nanoparticles is the core–shell structure exemplified by superparamagnetic iron oxide nanoparticles (SPIONs) grafted with dense, well-hydrated polymer shells used for biomedical magnetic imaging and therapy. Densely grafted polymer chains form a polymer brush, yielding a highly repulsive barrier to the formation of a protein corona via nonspecific particle–protein interactions. However, recent studies showed that the abundant blood serum protein albumin interacts with dense polymer brush-grafted SPIONs. Herein, we use isothermal titration calorimetry to characterize the nonspecific interactions between human serum albumin, human serum immunoglobulin G, human transferrin, and hen egg lysozyme with monodisperse poly(2-alkyl-2-oxazoline)-grafted SPIONs with different grafting densities and core sizes. These particles show similar protein interactions despite their different “stealth” capabilities in cell culture. The SPIONs resist attractive interactions with lysozymes and transferrins, but they both show a significant exothermic enthalpic and low exothermic entropic interaction with low stoichiometry for albumin and immunoglobulin G. Our results highlight that protein size, flexibility, and charge are important to predict protein corona formation on polymer brush-stabilized nanoparticles.

A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover.

A detailed understanding of the mechanisms underlying the capacity of a virus to break the species barrier is crucial for pathogen surveillance and control. New World (NW) mammarenaviruses constitute a diverse group of rodent-borne pathogens that includes several causative agents of severe viral hemorrhagic fever in humans. The ability of the NW mammarenaviral attachment glycoprotein (GP) to utilize human transferrin receptor 1 (hTfR1) as a primary entry receptor plays a key role in dictating zoonotic potential. The recent isolation of Tacaribe and lymphocytic choriominingitis mammarenaviruses from host-seeking ticks provided evidence for the presence of mammarenaviruses in arthropods, which are established vectors for numerous other viral pathogens. Here, using next generation sequencing to search for other mammarenaviruses in ticks, we identified a novel replication-competent strain of the NW mammarenavirus Tamiami (TAMV-FL), which we found capable of utilizing hTfR1 to enter mammalian cells. During isolation through serial passaging in mammalian immunocompetent cells, the quasispecies of TAMV-FL acquired and enriched mutations leading to the amino acid changes N151K and D156N, within GP. Cell entry studies revealed that both substitutions, N151K and D156N, increased dependence of the virus on hTfR1 and binding to heparan sulfate proteoglycans. Moreover, we show that the substituted residues likely map to the sterically constrained trimeric axis of GP, and facilitate viral fusion at a lower pH, resulting in viral egress from later endosomal compartments. In summary, we identify and characterize a naturally occurring TAMV strain (TAMV-FL) within ticks that is able to utilize hTfR1. The TAMV-FL significantly diverged from previous TAMV isolates, demonstrating that TAMV quasispecies exhibit striking genetic plasticity that may facilitate zoonotic spillover and rapid adaptation to new hosts.

A brain penetrant progranulin biotherapeutic rescues lysosomal and inflammatory phenotypes in the brain of GRN knockout mice

AbstractBackgroundHeterozygous loss of function (LOF) mutations in GRN cause frontotemporal dementia (FTD), a neurodegenerative disorder associated with lysosomal dysfunction, TDP‐43 pathology and inflammation in the CNS. Additionally, homozygous LOF mutations cause neuronal ceroid lipofuscinosis, a lysosomal storage disorder. GRN encodes progranulin (PGRN), a soluble protein that is most abundantly expressed in microglia, where it localizes to lysosomes and can undergo secretion. Although the precise function of PGRN is unknown, growing evidence suggests that it regulates lysosomal function, inflammatory responses and promotes neuronal survival. Accordingly, PGRN LOF models are associated with lysosomal defects, hyperinflammatory responses, and decreased neuronal viability, both in vitro and in vivo. Because GRN‐FTD patients exhibit reduced levels of PGRN in biofluids and tissues, including the brain, a protein replacement therapy analogous to enzyme replacement therapy and capable of crossing the blood brain barrier (BBB) more efficiently may represent a powerful approach to slow or prevent disease progression.MethodHere we describe a novel therapeutic for increasing brain penetrance of PGRN, referred to as Protein Transport Vehicle (PTV):PGRN. PTV:PGRN consists of recombinant human PGRN fused to a modified Fc domain engineered to bind to the human transferrin receptor (huTfR), thus facilitating receptor‐mediated transcytosis across the BBB.ResultWe found that extracellular applications of PTV:PGRN rescue a range of Grn KO cell phenotypes in vitro, including lysosomal proteolysis and alteration in the levels of bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid involved in lysosomal lipid catabolism. Intravenously administered PTV:PGRN showed increased acute brain exposure in huTfR knock‐in (TfRmu/hu) mice relative to a regular Fc:PGRN fusion that does not bind to huTfR. Importantly, low doses of PTV:PGRN, but not Fc:PGRN, fully corrected lysosomal lipid alterations, including BMP deficiency, as well as inflammatory markers in the brain of Grn KO x TfRmu/hu mice.ConclusionOur data suggest that PTV:PGRN may represent a viable therapeutic strategy for the treatment of GRN‐FTD and potentially other disorders associated with PGRN deficiency.

Transferrin improved the generation of cardiomyocyte from human pluripotent stem cells for myocardial infarction repair

Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold great promise for the repair of the injured heart, but optimal cell production in a fully chemically defined and cost-effective system is essential for the efficacy and safety of cell transplantation therapies. In this study, we provided a simple and efficient strategy for cardiac differentiation from hPSCs and performed functional evaluation in a rat model of myocardial infarction. Using a chemically defined medium including four components, recombinant human albumin, ascorbic acid, human transferrin, and RPMI 1640, we developed a manageable and cost-effective protocol for robust generation of CMs from hPSCs. Interestingly, the addition of transferrin helped hPSCs to transit from TeSR-E8 medium to the simple cardiac differentiation medium and successfully initiated mesoderm differentiation without significant cell death. The CM generation efficiency was up to 85% based on cTnT expression. We performed transcriptome profiling from differentiation day 0 to 35, and characterized interesting dynamic change of cardiac genes. CMs derived from transferrin-supplemented simple medium have similar transcriptome and the maturation level compared to those generated in B27 minus insulin medium as well as their in vivo counterparts. Importantly, after transplantation, hPSC-derived CMs survived in the infarcted rat heart, significantly improved the physiological function and reduced fibrosis. Our study offers an easy-to-use and cost-effective method for cardiac differentiation and facilitates the translational application of hPSC-derived CMs for heart repair.

Tuning the binding interface between Machupo virus glycoprotein and human transferrin receptor.

Machupo virus, known to cause hemorrhagic fevers, enters human cells via binding with its envelope glycoprotein to transferrin receptor 1 (TfR). Similarly, the receptor interactions have been explored in biotechnological applications as a molecular system to ferry therapeutics across the cellular membranes and through the impenetrable blood–brain barrier that effectively blocks any such delivery into the brain. Study of the experimental structure of Machupo virus glycoprotein 1 (MGP1) in complex with TfR and glycoprotein sequence homology has identified some residues at the interface that influence binding. There are, however, no studies that have attempted to optimize the binding potential between MGP1 and TfR. In pursuits for finding therapeutic solutions for the New World arenaviruses, and to gain a greater understanding of MGP1 interactions with TfR, it is crucial to understand the structure–sequence relationship driving the interface formation. By displaying MGP1 on yeast surface we have examined the contributions of individual residues to the binding of solubilized ectodomain of TfR. We identified MGP1 binding hot spot residues, assessed the importance of posttranslational N‐glycan modifications, and used a selection with random mutagenesis for affinity maturation. We show that the optimized MGP1 variants can bind more strongly to TfR than the native MGP1, and there is an MGP1 sequence that retains binding in the absence of glycosylation, but with the addition of further amino acid substitutions. The engineered variants can be used to probe cellular internalization or the blood‐brain barrier crossing to achieve greater understanding of TfR mediated internalization.

Non-Human Primate Blood-Brain Barrier and In Vitro Brain Endothelium: From Transcriptome to the Establishment of a New Model.

The non-human primate (NHP)-brain endothelium constitutes an essential alternative to human in the prediction of molecule trafficking across the blood–brain barrier (BBB). This study presents a comparison between the NHP transcriptome of freshly isolated brain microcapillaries and in vitro-selected brain endothelial cells (BECs), focusing on important BBB features, namely tight junctions, receptors mediating transcytosis (RMT), ABC and SLC transporters, given its relevance as an alternative model for the molecule trafficking prediction across the BBB and identification of new brain-specific transport mechanisms. In vitro BECs conserved most of the BBB key elements for barrier integrity and control of molecular trafficking. The function of RMT via the transferrin receptor (TFRC) was characterized in this NHP-BBB model, where both human transferrin and anti-hTFRC antibody showed increased apical-to-basolateral passage in comparison to control molecules. In parallel, eventual BBB-related regional differences were Investig.igated in seven-day in vitro-selected BECs from five brain structures: brainstem, cerebellum, cortex, hippocampus, and striatum. Our analysis retrieved few differences in the brain endothelium across brain regions, suggesting a rather homogeneous BBB function across the brain parenchyma. The presently established NHP-derived BBB model closely mimics the physiological BBB, thus representing a ready-to-use tool for assessment of the penetration of biotherapeutics into the human CNS.