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Summary: Renal transporters play critical roles in predicting potential drug-drug interactions. However, current in vitro models often fail to adequately express these transporters, particularly solute carrier proteins, including organic anion transporters (OAT1/3), and organic cation transporter 2 (OCT2). Here, we developed a hiPSC-derived kidney organoids-based proximal tubule-on-chip (OPTC) model that emulates in vivo renal physiology to assess transporter function. Compared to chips based on immortalized cells, OPTC derived from the two most commonly used differentiation protocols exhibited significant improvement in expression level and polarity of OAT1/3 and OCT2. Hence, the OPTC demonstrates enhanced functionality in efflux and uptake assessments, and nephrotoxicity. Furthermore, these functionalities are diminished upon adding inhibitors during substrate-inhibitor interactions, which were closer to in vivo observations. Overall, these results support that OPTC can reliably assess the role of renal transporters in drug transport and nephrotoxicity, paving the way for personalized models to assess renal transport and disease modeling.

Summary: The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these “supraG4zymes” enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments.

Todays Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrows products and processes. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the worlds grand challenges.

Summary: Severe acute pancreatitis (AP) is associated with a high mortality rate. Cold-inducible RNA binding protein (CIRP) can be released from cells in inflammatory conditions and extracellular CIRP acts as a damage-associated molecular pattern. This study aims to explore the role of CIRP in the pathogenesis of AP and evaluate the therapeutic potential of targeting extracellular CIRP with X-aptamers. Our results showed that serum CIRP concentrations were significantly increased in AP mice. Recombinant CIRP triggered mitochondrial injury and ER stress in pancreatic acinar cells. CIRP−/− mice suffered less severe pancreatic injury and inflammatory responses. Using a bead-based X-aptamer library, we identified an X-aptamer that specifically binds to CIRP (XA-CIRP). Structurally, XA-CIRP blocked the interaction between CIRP and TLR4. Functionally, it reduced CIRP-induced pancreatic acinar cell injury in vitro and L-arginine-induced pancreatic injury and inflammation in vivo. Thus, targeting extracellular CIRP with X-aptamers may be a promising strategy to treat AP.

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.

The rational design of Ribonucleic acid (RNA) molecules is crucial for advancing therapeutic applications, synthetic biology, and understanding the fundamental principles of life. Traditional RNA design methods have predominantly focused on secondary structure-based sequence design, often neglecting the intricate and essential tertiary interactions. We introduce R3Design, a tertiary structure-based RNA sequence design method that shifts the paradigm to prioritize tertiary structure in the RNA sequence design. R3Design significantly enhances sequence design on native RNA backbones, achieving high sequence recovery and Macro-F1 score, and outperforming traditional secondary structure-based approaches by substantial margins. We demonstrate that R3Design can design RNA sequences that fold into the desired tertiary structures by validating these predictions using advanced structure prediction models. This method, which is available through standalone software, provides a comprehensive toolkit for designing, folding, and evaluating RNA at the tertiary level. Our findings demonstrate R3Design's superior capability in designing RNA sequences, which achieves around $44\%$ in terms of both recovery score and Macro-F1 score in multiple datasets. This not only denotes the accuracy and fairness of the model but also underscores its potential to drive forward the development of innovative RNA-based therapeutics and to deepen our understanding of RNA biology., (© The Author(s) 2024. Published by Oxford University Press.)

Summary: Nucleic acid-binding polymers can have anti-inflammatory properties and beneficial effects in animal models of infection, trauma, cancer, and autoimmunity. PAMAM G3, a polyamidoamine dendrimer, is fully cationic bearing 32 protonable surface amines. However, while PAMAM G3 treatment leads to improved outcomes for mice infected with influenza, at risk of cancer metastasis, or genetically prone to lupus, its administration can lead to serosal inflammation and elevation of biomarkers of liver and kidney damage. Variants with reduced density of cationic charge through the interspersal of hydroxyl groups were evaluated as potentially better-tolerated alternatives. Notably, the variant PAMAM G3 50:50, similar in size as PAMAM G3 but with half the charge, was not toxic in cell culture, less associated with weight loss or serosal inflammation after parenteral administration, and remained effective in reducing glomerulonephritis in lupus-prone mice. Identification of such modified scavengers should facilitate their development as safe and effective anti-inflammatory agents.

Renal transporters play critical roles in predicting potential drug-drug interactions. However, current in vitro models often fail to adequately express these transporters, particularly solute carrier proteins, including organic anion transporters (OAT1/3), and organic cation transporter 2 (OCT2). Here, we developed a hiPSC-derived kidney organoids-based proximal tubule-on-chip (OPTC) model that emulates in vivo renal physiology to assess transporter function. Compared to chips based on immortalized cells, OPTC derived from the two most commonly used differentiation protocols exhibited significant improvement in expression level and polarity of OAT1/3 and OCT2. Hence, the OPTC demonstrates enhanced functionality in efflux and uptake assessments, and nephrotoxicity. Furthermore, these functionalities are diminished upon adding inhibitors during substrate-inhibitor interactions, which were closer to in vivo observations. Overall, these results support that OPTC can reliably assess the role of renal transporters in drug transport and nephrotoxicity, paving the way for personalized models to assess renal transport and disease modeling., Competing Interests: R.Y., C.M., R.B.S., K.F., T.A., M.T., and R.N. are inventors on JP patent application no. 2024–73489 “Construction of proximal tubules micro-physiological system”., (© 2024 The Author(s).)

Summary: Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3′ interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.

Summary: ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular responses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensitizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and necroptosis. We also decrypted a temporally shifted amplitude- and frequency-modulated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Summary: Mitochondrial biogenesis is a cell response to external stimuli which is generally believed to suppress apoptosis. However, during the process of apoptosis, whether mitochondrial biogenesis occurs in the early stage of the apoptotic cells remains unclear. To address this question, we constructed the COX8-EGFP-ACTIN-mCherry HeLa cells with recombinant fluorescent proteins respectively tagged on the nucleus and mitochondria and monitored the mitochondrial changes in the living cells exposed to gamma-ray radiation. Besides in situ detection of mitochondrial fluorescence changes, we also examined the cell viability, nuclear DNA damage, reactive oxygen species (ROS), mitochondrial superoxide, citrate synthase activity, ATP, cytoplasmic and mitochondrial calcium, mitochondrial mass, mitochondrial morphology, and protein expression related to mitochondrial biogenesis, as well as the apoptosis biomarkers. As a result, we confirmed that significant mitochondrial biogenesis took place preceding the radiation-induced apoptosis, and it was closely correlated with the apoptotic cells at late stage. The involved mechanism was also discussed.

Summary: Unfolding followed by fibrillation of insulin even in the presence of various excipients grappled with restricted clinical application. Thus, there is an unmet need for better thermostable, nontoxic molecules to preserve bioactive insulin under varying physiochemical perturbations. In search of cross-amyloid inhibitors, prion-derived tetrapeptide library screening reveals a consensus V(X)YR motif for potential inhibition of insulin fibrillation. A tetrapeptide VYYR, isosequential to the β2-strand of prion, effectively suppresses heat- and storage-induced insulin fibrillation and maintains insulin in a thermostable bioactive form conferring adequate glycemic control in mouse models of diabetes and impedes insulin amyloidoma formation. Besides elucidating the critical insulin-IS1 interaction (R4 of IS1 to the N24 insulin B-chain) by nuclear magnetic resonance spectroscopy, we further demonstrated non-canonical dimer-mediated conformational trapping mechanism for insulin stabilization. In this study, structural characterization and preclinical validation introduce a class of tetrapeptide toward developing thermostable therapeutically relevant insulin formulations.

Summary: Lanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

The distinct folding accompanied by its polymorphic character renders DNA G-quadruplexes promising biomolecular building blocks to construct novel DNA-based and supramolecular assemblies. However, the highly polar nature of DNA limits the use of G-quadruplexes to water as a solvent. In addition, the archetypical G-quadruplex fold needs to be stabilized by metal-cations, which is usually a potassium ion. Here, we show that a noncovalent PEGylation process enabled by electrostatic interactions allows the first metal-free G-quadruplexes in organic solvents. Strikingly, incorporation of an iron-containing porphyrin renders the self-assembled metal-free G-quadruplex catalytically active in organic solvents. Hence, these "supraG4zymes" enable DNA-based catalysis in organic media. The results will allow the broad utilization of DNA G-quadruplexes in nonaqueous environments., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s).)

Although natural or artificial modified pyrimidine nucleobases represent important molecules with valuable properties as constituents of DNA and RNA, no systematic analyses of the structural aspects of bromo derivatives of cytosine have appeared so far in the literature. In view of the biochemical and pharmaceutical relevance of these compounds, six different crystals containing proton-transfer derivatives of 5-bromocytosine are prepared and analyzed in the solid-state by single crystal X-ray diffraction. All six compounds are organic salts, with proton transfer occurring to the Nimino atom of the pyridine ring. Experimental results are then complemented with Hirshfeld surface analysis to quantitively evaluate the contribution of different intermolecular interactions in the crystal packing. Furthermore, theoretical calculations, based on different arrangements of molecules extracted from the crystal structure determinations, are carried out to analyze the formation mechanism of halogen bonds (XBs) in these compounds and provide insights into the nature and strength of the observed interactions. The results show that the supramolecular architectures of the six molecular salts involve extensive classical intermolecular hydrogen bonds. However, in all but one proton-transfer adducts, weak to moderate XBs are revealed by C–Br…O short contacts between the bromine atom in the fifth position, which acts as XB donor (electron acceptor). Moreover, the lone pair electrons of the oxygen atom of adjacent pyrimidine nucleobases and/or counterions or water molecules, which acts as XB acceptor (electron donor).

Severe acute pancreatitis (AP) is associated with a high mortality rate. Cold-inducible RNA binding protein (CIRP) can be released from cells in inflammatory conditions and extracellular CIRP acts as a damage-associated molecular pattern. This study aims to explore the role of CIRP in the pathogenesis of AP and evaluate the therapeutic potential of targeting extracellular CIRP with X-aptamers. Our results showed that serum CIRP concentrations were significantly increased in AP mice. Recombinant CIRP triggered mitochondrial injury and ER stress in pancreatic acinar cells. CIRP -/- mice suffered less severe pancreatic injury and inflammatory responses. Using a bead-based X-aptamer library, we identified an X-aptamer that specifically binds to CIRP (XA-CIRP). Structurally, XA-CIRP blocked the interaction between CIRP and TLR4. Functionally, it reduced CIRP-induced pancreatic acinar cell injury in vitro and L-arginine-induced pancreatic injury and inflammation in vivo . Thus, targeting extracellular CIRP with X-aptamers may be a promising strategy to treat AP., Competing Interests: The authors reported no conflict of interest in this study., (© 2023 The Author(s).)

Engineering materials using supramolecular principles enables generalizable and modular platforms that have tunable chemical, mechanical, and biological properties. Applying this bottom-up, molecular engineering-based approach to therapeutic design affords unmatched control of emergent properties and functionalities. In preparing responsive materials for biomedical applications, the dynamic character of typical supramolecular interactions facilitates systems that can more rapidly sense and respond to specific stimuli through a fundamental change in material properties or characteristics, as compared to cases where covalent bonds must be overcome. Several supramolecular motifs have been evaluated toward the preparation of 'smart' materials capable of sensing and responding to stimuli. Triggers of interest in designing materials for therapeutic use include applied external fields, environmental changes, biological actuators, applied mechanical loading, and modulation of relative binding affinities. In addition, multistimuli-responsive routes can be realized that capture combinations of triggers for increased functionality. In sum, supramolecular engineering offers a highly functional strategy to prepare responsive materials. Future development and refinement of these approaches will improve precision in material formation and responsiveness, seek dynamic reciprocity in interactions with living biological systems, and improve spatiotemporal sensing of disease for better therapeutic deployment. [ABSTRACT FROM AUTHOR]

Due to inherent complexity, incompatibility, and variability in living cell systems, biomolecular engineering faces significant obstacles. To find novel solutions to these issues, researchers have turned to cell-free synthetic biology (CFSB), a relatively young field of study. Biochemical processes can be triggered in vitro through cell-free synthesis, providing a wider range of options for biomolecular engineering. Here, we provide a survey of recent advances in cell-free synthesis. These have sparked innovative studies in areas including the synthesis of complex proteins, incorporation of unnatural amino acids, precise post-translational modifications, high-throughput workflow, and synthetic biomolecular network regulation. CFSB has transformed the studies of biological machinery in a profound and practical way for versatile biomolecular engineering applications., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Nucleic acid-binding polymers can have anti-inflammatory properties and beneficial effects in animal models of infection, trauma, cancer, and autoimmunity. PAMAM G3, a polyamidoamine dendrimer, is fully cationic bearing 32 protonable surface amines. However, while PAMAM G3 treatment leads to improved outcomes for mice infected with influenza, at risk of cancer metastasis, or genetically prone to lupus, its administration can lead to serosal inflammation and elevation of biomarkers of liver and kidney damage. Variants with reduced density of cationic charge through the interspersal of hydroxyl groups were evaluated as potentially better-tolerated alternatives. Notably, the variant PAMAM G3 50:50, similar in size as PAMAM G3 but with half the charge, was not toxic in cell culture, less associated with weight loss or serosal inflammation after parenteral administration, and remained effective in reducing glomerulonephritis in lupus-prone mice. Identification of such modified scavengers should facilitate their development as safe and effective anti-inflammatory agents., Competing Interests: Duke University has applied for patents on the strategy to reduce inflammation via nucleic acid scavengers. Lyra Olson, Nicole Hunter, Rachel Rempel, and Bruce Sullenger are listed as inventors on such patents., (© 2022 The Author(s).)

Abstract: The engineering of biological systems requires a set of molecular tools that can be predictably applied to the design of sophisticated genetic systems. Recent advances in the field of RNA synthetic biology, particularly in the design of synthetic regulatory RNAs for the static and dynamic control of gene expression, have increased our ability to efficiently build various biological systems capable of performing programmed cellular behaviors. Furthermore, implementing these synthetic regulatory RNAs in biological systems highlights the potential for designing synthetic cell systems for chemical synthesis, environmental, agricultural, and medical applications. In this paper, we review current developments in synthetic regulatory RNAs for the static and dynamic control of gene expression and the potential applications of these tools. [Copyright &y& Elsevier]

Abstract: Synthetic Biology offers qualitatively new perspectives on the benefits of industrially harnessed biological processes. The ability to modify and reprogramme natural biology increases the scope of tailored bioprocesses and yields attractive prospects beyond conventional Biotechnology. The present review summarises the major achievements and categorises them according to a hierarchy of system levels. Similar structures are known in the engineering sciences and might prove useful for the future development of Synthetic Biology. The hierarchy encompasses several levels of detail. Biological (macro-)molecules present the most detailed level (parts), followed by compartmentalised or non-compartmentalised modules (devices). In the next level, parts and devices are combined into functional cells and further into cellular communities. The manifold interactions between biological entities of the same hierarchical level or between different levels are accounted for by networks, primarily metabolic pathways and regulatory circuits. Networks of different types are represented as a superordinate hierarchical level that achieves full system integration. On all these levels, extensive and sound scientific foundations exist regarding experimental but also theoretical methods. These have led to diverse manifestations of Synthetic Biology on the parts and devices levels. Investigations involving synthetic components on the systems scale represent the most difficult and remain limited in number. A main challenge lies with the quantitative prediction of interactions between different entities across different scales. Systems-theoretical approaches provide important tools to analyse complex biological behaviour and can support the design of artificial biological systems. A promising strategy is seen in an efficient modularisation that reduces biological systems to a limited set of functional modules with well-characterised interfaces. For the design of synthetic biological systems the interactions across these interfaces should be standardised to reduce complexity. Yet, the identification of modules and standardised interaction routes remains a non-trivial problem. Furthermore, an appropriate platform that efficiently describes replication and evolutionary processes has to be developed in order to extend the achievements of Synthetic Biology into designed biological processes. [Copyright &y& Elsevier]

Abstract: A review of current processing practices in preparation of recombinant single chain antibody fragments is presented. Single chain antibody fragments which are superior to their Fab and IgG counterparts due to their higher affinity for target antigens while imposing minimal antigenicity in recipient hosts, have sparked breakthroughs in immunology and the medical field at large. The rapidly increasing market demand for pure single chain antibodies for research and therapeutic applications, necessitates viable manufacture routes that can produce large amounts of these antibodies efficiently and as cheaply as possible. Medium- to high-producing expression systems reported for recombinant single chain antibody production are reviewed, and their reported or potential success for efficient commercial-viable preparation of pure antibodies discussed. The effects of expression host system choice on product molecular constraints, ease of processing, and flowsheet design and scale-up are compared. It is concluded that there is no unique host system that can consistently yield high expression levels for a wide range of single chain antibodies; instead, product sequence and end application often dictate the optimum choice of expression host. Irrespective of host systems, adequate a priori design and engineering of the molecular construct supported by good biophysical understanding of the single chain fragment molecules, is a crucial pre-requisite for improved product stability and downstream recovery, which will favourably impact final product yield and functionality. [Copyright &y& Elsevier]