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Abstract Ultra-tight binding is usually observed for proteins associating with rigidified molecules. Previously, we demonstrated that femtomolar binders derived from the Armadillo repeat proteins (ArmRPs) can be designed to interact very tightly with fully flexible peptides. Here we show for ArmRPs with four and seven sequence-identical internal repeats that the peptide-ArmRP complexes display conformational dynamics. These dynamics stem from transient breakages of individual protein-residue contacts that are unrelated to overall unbinding. The labile contacts involve electrostatic interactions. We speculate that these dynamics allow attaining very high binding affinities, since they reduce entropic losses. Importantly, only NMR techniques can pick up these local events by directly detecting conformational exchange processes without complications from changes in solvent entropy. Furthermore, we demonstrate that the interaction surface of the repeat protein regularizes upon peptide binding to become more compatible with the peptide geometry. These results provide novel design principles for ultra-tight binders.

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized.Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy.Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50 nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25 nm. Confocal imaging unequivocally illustrated MitoView 633’s specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells.Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.

Human induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) are based on ground-breaking technology that has significantly impacted cardiovascular research. They provide a renewable source of human cardiomyocytes for a variety of applications including in vitro disease modeling and drug toxicity testing. Cardiac calcium regulation plays a critical role in the cardiomyocyte and is often dysregulated in cardiovascular disease. Due to the limited availability of human cardiac tissue, calcium handling and its regulation have most commonly been studied in the context of animal models. hiPSC-CMs can provide unique insights into human physiology and pathophysiology, although a remaining limitation is the relative immaturity of these cells compared to adult cardiomyocytes Therefore, this field is rapidly developing techniques to improve the maturity of hiPSC-CMs, further establishing their place in cardiovascular research. This review briefly covers the basics of cardiomyocyte calcium cycling and hiPSC technology, and will provide a detailed description of our current understanding of calcium in hiPSC-CMs.

OBJECTIVES/GOALS: We will use control- and DMD-engineered heart tissues to better model and investigate DMD cardiomyopathy. We will primarily assess cardiac calcium handling, mitochondrial function, and mitochondrial calcium handling, as calcium regulation and mitochondrial function are known to be affected in DMD. METHODS/STUDY POPULATION: We will use patient-derived stem cells, differentiated into cardiomyocytes in bioprinted 3D heart tissue muscle chambers to better model DMD cardiomyopathy. We will look at calcium handling and general mitochondrial function, as well as mitochondrial calcium handling, using a novel multifunctional genetic probe I previously developed allowing for simultaneous observation of cytosolic and mitochondrial calcium in real time. Optical mapping will also be used for tissue-level analysis. We will establish the functional differences at baseline, and then progress heart failure in the tissues to see how the abnormalities seen in the DMD tissues may get worse. Finally, we will investigate the effects of early restoration of dystrophin function on the effects of DMD cardiomyopathy development. RESULTS/ANTICIPATED RESULTS: We anticipate that DMD tissues will show more irregular/abnormal calcium handling, as seen in 2D hiPSC-CMs, as well as disruptions to mitochondrial function and ultrastructural development, as well as a decreased synchronization between cytosolic and mitochondrial calcium dynamics. We anticipate that these abnormalities will be exacerbated as the disease state progresses, but at least partially ameliorated with the restoration of dystrophin function. DISCUSSION/SIGNIFICANCE: DMD is a fatal disease with no known cure. Patients develop heart failure in their teens and die in their 20s, so any new insight that may prolong life and improve quality of life for patients is drastically needed. This would be the most accurate preclinical model of DMD cardiomyopathy to date and would investigate yet-untapped aspects of the disease state.

The V3-crown of the HIV-1 envelope protein largely elicits non-neutralizing antibodies. Here, the authors show that the V3-crown can be targeted by broadly neutralizing designed ankyrin repeat proteins recognizing two conformations one of which resembles CCR5- bound V3.

HER2 acts an oncogenic driver in numerous cancers. Here, the authors design an anti-HER2 biparatopic and tetravalent IgG fusion with inhibitory effects in a xenograft model.

Viral gene therapy can be limited by the efficacy of virion sequestration, immune responses and the silencing of genetic payloads. Here the authors engineer an advenovirus protein coat which shields the virion from the immune system while targeting cancer cells.

TDP-43 aggregation is observed in amyotrophic lateral sclerosis. Here the authors combine X-ray crystallography, nuclear magnetic resonance and electron microscopy studies and show that physiological oligomerization of TDP-43 is mediated through its N-terminal domain, which forms functional and dynamic oligomers antagonizing pathologic aggregation.

Summary: Bacteriophages provide excellent tools for diagnostics, remediation, and targeted microbiome manipulation, yet isolating viruses with suitable host specificity remains challenging. Using Listeria phage PSA, we present a synthetic biology blueprint for host-range engineering through targeted modification of serovar-specific receptor binding proteins (RBPs). We identify Gp15 as the PSA RBP and construct a synthetic phage library featuring sequence-randomized RBPs, from which host range mutants are isolated and subsequently integrated into a synthetic, polyvalent phage with extended host range. To enable rational design of chimeric RBPs, we determine the crystal structure of the Gp15 receptor-binding carboxyl terminus at 1.7-Å resolution and employ bioinformatics to identify compatible, prophage-encoded RBPs targeting different Listeria serovars. Structure-guided design enables exchange of heterologous RBP head, neck, or shoulder domains to generate chimeric phages with predictable and extended host ranges. These strategies will facilitate the development of phage biologics based on standardized virus scaffolds with tunable host specificities. : Receptor binding proteins (RBPs) are key determinants of bacteriophage host range. Dunne et al. solve the RBP crystal structure of Listeria phage PSA and combine synthetic biology with structure-guided approaches to re-program RBP specificity. Using the temperate, narrow host range PSA scaffold, they construct virulent phages with extended and defined host specificities. Keywords: bacteriophage, listeria, phage engineering, tail spike, receptor binding protein, X-ray crystallography, host range, directed evolution, synthetic biology, chimeric phage

Meningiomas are primary tumors of the central nervous system with high recurrence. It has been reported that somatostatin receptor 2 (SSTR2) is highly expressed in most meningiomas, but there is no effective targeted therapy approved to control meningiomas. This study aimed to develop and evaluate an anti-SSTR2 antibody–drug conjugate (ADC) to target and treat meningiomas. The meningioma targeting, circulation stability, toxicity, and anti-tumor efficacy of SSTR2 ADC were evaluated using cell lines and/or an intracranial xenograft mouse model. The flow cytometry analysis showed that the anti-SSTR2 mAb had a high binding rate of >98% to meningioma CH157-MN cells but a low binding rate of 50 value of

Background Oxidative stress–mediated Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of cardiac ion channels has emerged as a critical contributor to arrhythmogenesis in cardiac pathology. However, the link between mitochondrial‐derived reactive oxygen species (mdROS) and increased CaMKII activity in the context of cardiac arrhythmias has not been fully elucidated and is difficult to establish experimentally. Methods and Results We hypothesize that pathological mdROS can cause erratic action potentials through the oxidation‐dependent CaMKII activation pathway. We further propose that CaMKII‐dependent phosphorylation of sarcolemmal slow Na+ channels alone is sufficient to elicit early afterdepolarizations. To test the hypotheses, we expanded our well‐established guinea pig cardiomyocyte excitation‐contraction coupling, mitochondrial energetics, and ROS‐induced‐ROS‐release model by incorporating oxidative CaMKII activation and CaMKII‐dependent Na+ channel phosphorylation in silico. Simulations show that mdROS mediated‐CaMKII activation elicits early afterdepolarizations by augmenting the late Na+ currents, which can be suppressed by blocking L‐type Ca2+ channels or Na+/Ca2+ exchangers. Interestingly, we found that oxidative CaMKII activation–induced early afterdepolarizations are sustained even after mdROS has returned to its physiological levels. Moreover, mitochondrial‐targeting antioxidant treatment can suppress the early afterdepolarizations, but only if given in an appropriate time window. Incorporating concurrent mdROS‐induced ryanodine receptors activation further exacerbates the proarrhythmogenic effect of oxidative CaMKII activation. Conclusions We conclude that oxidative CaMKII activation–dependent Na channel phosphorylation is a critical pathway in mitochondria‐mediated cardiac arrhythmogenesis.