We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a ...sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes.
Chimeric proteins, including bispecific antibodies, are biological tools with therapeutic applications. Genetic fusion and ligation methods allow the creation of N-to-C and C-to-N fused recombinant ...proteins, but not unnaturally linked N-to-N and C-to-C fusion proteins. This protocol describes a simple procedure for the production of such chimeric proteins, starting from correctly folded proteins and readily available peptides. By equipping the N terminus or C terminus of the proteins of interest with a set of click handles using sortase A, followed by a strain-promoted click reaction, unnatural N-to-N and C-to-C linked (hetero) fusion proteins are established. Examples of proteins that have been conjugated via this method include interleukin-2, interferon-α, ubiquitin, antibodies and several single-domain antibodies. If the peptides, sortase A and the proteins of interest are in hand, the unnaturally N-to-N and C-to-C fused proteins can be obtained in 3-4 d.
Pectus excavatum is the most common congenital chest wall deformity. Customised silicone implants have been used to camouflage this deformity with good short-term outcomes. In the long term, ...permanent implants have a significant risk of capsular contracture, migration and extrusion. Scaffold-guided tissue engineering provides an alternative autologous solution which avoids issues associated with permanent implants. We implanted a 3D-printed, custom-made, biodegradable and highly porous scaffold filled with autologous fat graft. We were able to sustain autologous fat in the construct. There was an excellent aesthetic outcome and the highly porous polycaprolactone implant was well tolerated by the patient. This case illustrates the first-in-human trial of soft tissue engineering to camouflage a pectus excavatum defect not reconstructable by conventional techniques.
Level of evidence: Level V, therapeutic study.
Building heating demand makes up about a third of final energy consumption in Germany. Increasing energy efficiency and switching to alternative heating technologies, e.g., based on renewable ...energies, plays a pivotal role in achieving greenhouse gas emission targets in the building sector. In developing meaningful and efficient policies such as incentive schemes, a thorough understanding of the building sector structures and heating systems is crucial. To approach the said matter, we couple an urban energy system platform to assess single building heat demands based on geometrically exact 3D building models (SimStadt) with a mixed-integer linear program (MILP) for cost-optimization of residential technology investment and operation (COMODO). We apply the novel bottom-up model framework to show the loss of heterogeneity in actual building geometries and their physical properties induced by using simplified approaches with archetypal buildings. In the analysis, 100 buildings of the same TABULA building type are simulated and optimized with the proposed model framework. We assess heterogeneity on several levels, including net energy demand, the choice of cost-optimal heating system, and final energy demand: for example, specific heating demand varies by 20% between the 100 case study buildings which would have hitherto considered to have equal demand. This spread shows that details such as building geometries are a critical factor in modeling energy systems on a local scale.
Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to
-acetylgalactosamine (GalNAc) ligands has become the primary strategy for ...hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.
Toward Community Standards and Software for Whole-Cell Modeling Waltemath, Dagmar; Pir, Pinar; Alaybeyoglu, Begum ...
IEEE transactions on bio-medical engineering/IEEE transactions on biomedical engineering,
10/2016, Letnik:
63, Številka:
10
Journal Article
Recenzirano
Odprti dostop
Objective: Whole-cell (WC) modeling is a promising tool for biological research, bioengineering, and medicine. However, substantial work remains to create accurate comprehensive models of complex ...cells. Methods: We organized the 2015 Whole-Cell Modeling Summer School to teach WC modeling and evaluate the need for new WC modeling standards and software by recoding a recently published WC model in the Systems Biology Markup Language. Results: Our analysis revealed several challenges to representing WC models using the current standards. Conclusion: We, therefore, propose several new WC modeling standards, software, and databases. Significance: We anticipate that these new standards and software will enable more comprehensive models.
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