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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).)

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).)

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).)

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).)

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., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

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., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

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., Competing Interests: The authors declare that they have no competing interests., (© 2021 The Author(s).)

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., Competing Interests: There is no conflict of interest., (© 2021 The Authors.)

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., Competing Interests: The authors declare no competing interests., (© 2020 The Authors.)