Fluorescence Resonance Energy Transfer Experiments Research Articles

Waterborne 2-component polyurethane coatings based on acrylic polyols with secondary alcohols

We report the effects of increasing the molecular weight of a secondary alcohol-based polyol in a two-component waterborne polyurethane (2 K-WB-PUR) formulation. The system consists of an acrylic polyol latex and a water-dispersible polyisocyanate (hmPIC, Basonat HW1000 from BASF). The polyols investigated incorporated hydroxypropyl methacrylate as a comonomer to introduce the secondary –OH groups, in contrast to typical polyols which have primary -OH groups. Three molecular weights of this polyol were prepared (Mn ≈ 5100, Ð = 2.4; 10,300, Ð = 2.7; or 12,900 g/mol, Ð = 3.6) with a uniform Tg (∼20 °C) and hydrodynamic diameter (dh) ≈ 120 nm. The polyol and polyisocyanate were mixed with a molar NCO:OH ratio of 1.3:1. The particle size of the mixed dispersion was monitored by dynamic light scattering. The 5 K sample remained stable in dispersion over 7 days whereas the higher molecular weight formulations flocculated after 1 day. Fourier Transform Infrared (FTIR) measurements revealed that all NCO groups were consumed within 15–23 h for all dispersed samples. Fluorescence resonance energy transfer (FRET) experiments were carried out on samples in the dispersed state as well as on films formed from these dispersions. Very little molecular level mixing was observed in the dispersed phase for the samples. In the films, a large extent of mixing was observed in the 5 K sample whereas the extents of mixing for the higher molecular weight samples were reduced. The FTIR measurements recorded as the films formed showed that the NCO consumption in the 5 K film (t1/2 = 56 h) was comparable to an analogous 2 K-WB-PUR formulation based on a 5 K primary alcohol from a previous study. Surprisingly, the NCO consumption in the higher molecular weight samples was faster (t1/2 = 31, 32 h). We found that the composition of the resulting films formed from higher molecular weight polyols contained a greater relative ratio of polyurea to polyurethane.

YAP upregulates AMPKα1 to induce cancer cell senescence

Yes-associated protein (YAP)—a major effector protein of the Hippo pathway— regulates cell proliferation, differentiation, apoptosis, and senescence. Amp-activated protein kinase (AMPK) is a key sensor that monitors cellular nutrient supply and energy status. Although YAP and AMPK are considered to regulate cellular senescence, it is still unclear whether AMPK is involved in YAP-regulated cellular senescence. Here, we found that YAP promoted AMPKα1 aggregation and localization around mitochondria by co-transfecting CFP-YAP and YFP-AMPKα1 plasmids. Subsequent live cell fluorescence resonance energy transfer (FRET) assay did not exhibit direct interaction between YAP and AMPKα1. FRET, Co-immunoprecipitation, and western blot experiments revealed that YAP directly bound to TEAD, enhancing the expression of AMPKα1 and p-AMPKα. Treatment with verteporfin inhibited YAP’s binding to TEAD and reversed the elevated expression of AMPKα1 in the cells overexpressing CFP-YAP. Verteporfin also reduced the proportion of AMPKα1 puncta in the cells co-expressing CFP-YAP and YFP-AMPKα1. In addition, the AMPKα1 puncta were demonstrated to inhibit cell viability, autophagy, and proliferation, and ultimately promote cell senescence. In conclusion, YAP binds to TEAD to upregulate AMPKα1 and promotes the formation of AMPKα1 puncta around mitochondria under the condition of co-expression of CFP-YAP and YFP-AMPKα1, in which AMPKα1 puncta lead to cellular senescence.

Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages.

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336ΔfliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demonstrated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co-transcription. The deletion mutant ΔfliCΔdinJ increased cell death and IL-1β secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1β secretion in cells infected with ΔfliCΔdinJ compared to cells infected with ΔfliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow-derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3-/- and Nlrc4-/- mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1β and IL-18 in vivo, contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflammasome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.

Structural basis for CFTR inhibition by CFTRinh-172

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.

Deep-LASI, single-molecule data analysis software

By avoiding ensemble averaging, single-molecule methods provide novel means of extracting mechanistic insights into function of material and molecules at the nanoscale. However, one of the big limitations is the vast amount of data required for analyzing and extracting the desired information, which is time-consuming and user dependent. Here, we introduce Deep-LASI, a software suite for the manual and automatic analysis of single-molecule traces, interactions, and the underlying kinetics. The software can handle data from one-, two- and three-color fluorescence data, and was particularly designed for the analysis of two- and three-color single-molecule fluorescence resonance energy transfer experiments. The functionalities of the software include: the registration of multiple-channels, trace sorting and categorization, determination of the photobleaching steps, calculation of fluorescence resonance energy transfer correction factors, and kinetic analyses based on hidden Markov modeling or deep neural networks. After a kinetic analysis, the ensuing transition density plots are generated, which can be used for further quantification of the kinetic parameters of the system. Each step in the workflow can be performed manually or with the support of machine learning algorithms. Upon reading in the initial data set, it is also possible to perform the remaining analysis steps automatically without additional supervision. Hence, the time dedicated to the analysis of single-molecule experiments can be reduced from days/weeks to minutes. After a thorough description of the functionalities of the software, we also demonstrate the capabilities of the software via the analysis of a previously published dynamic three-color DNA origami structure fluctuating between three states. With the drastic time reduction in data analysis, new types of experiments become realistically possible that complement our currently available palette of methodologies for investigating the nanoworld.

Cbl-b negatively regulates TLR/MyD88-mediated anti-Toxoplasma gondii immunity.

Toxoplasma gondii is an important intracellular protozoan, which needs to exploit host nutrition for successful parasitism. We previously reported that Casitas B-lineage lymphoma-b (Cbl-b) was screened as a host dependency factor of T. gondii by using lentiviral CRISPR-Cas9-single guide RNA (sgRNA) libraries. Furthermore, the detailed mechanism of Cbl-b being required by T. gondii infection was explored in this study. The proliferation of T. gondii was found to be significantly inhibited in Cbl-b knockdown cell lines, and the Cbl-b expression level increased with prolonged T. gondii infection, while the MyD88 level was significantly decreased in the T. gondii infection group. Toll-like receptor (TLR)/MyD88 is a conserved innate cellular immune signaling pathway against pathogens infection. Cbl-b was found to interact with MyD88 and mediate MyD88 ubiquitination in the fluorescence resonance energy transfer and co-immunoprecipitation experiments. A Cbl-b knockout (KO) C57BL/6J lineage was then constructed and, together with the wild-type (WT) mice, was infected with the same amount of T. gondii tachyzoites of ME49 strain. At 13 days post infection, all the mice in the WT group died, while 80% of the mice in the Cbl-b KO group still survived, even to the end of the experiment. The parasitic burden in the liver, lung, and brain of the Cbl-b KO mice was significantly lower than that of the WT mice (P < 0.05). In Cbl-b KO infected mice, the percentage of B cells was higher, whereas that of macrophages was lower, and the interferon-γ and interleukin-6 levels in the serum were higher than that in the WT infected mice; the difference was significant (P < 0.05). Furthermore, when host cells were infected by T. gondii, host's Cbl-b was found to interact with MyD88 to ubiquitinate MyD88 for ribosome-dependent degradation. Therefore, the host's innate immunity against T. gondii through the TLR/MyD88 pathway was negatively regulated by Cbl-b.IMPORTANCEThis is the first report that a human E3 ubiquitin ligase, Casitas B-lineage lymphoma proto-oncogene B (Cbl-b), functions as a host dependency factor for the intracellular protozoan Toxoplasma gondii and the mechanism for how T. gondii infection inhibits the TLR/MyD88 innate immunity pathway through MyD88 degradation mediated by Cbl-b. This finding is an impactful contribution for understanding the host cell immunity against T. gondii infection.

Exploring Intra- and Inter-Regional Interactions in the IDP α-Synuclein Using smFRET and MD Simulations.

Theoretical concepts from polymer physics are often used to describe intrinsically disordered proteins (IDPs). However, amino acid interactions within and between regions of the protein can lead to deviations from typical polymer scaling behavior and even to short-lived secondary structures. To investigate the key interactions in the dynamic IDP α-synuclein (αS) at the amino acid level, we conducted single-molecule fluorescence resonance energy transfer (smFRET) experiments and coarse-grained molecular dynamics (CG-MD) simulations. We find excellent agreement between experiments and simulations. Our results show that a physiological salt solution is a good solvent for αS and that the protein is highly dynamic throughout its entire chain, with local intra- and inter-regional interactions leading to deviations from global scaling. Specifically, we observe expansion in the C-terminal region, compaction in the NAC region, and a slightly smaller distance between the C- and N-termini than expected. Our simulations indicate that the compaction in the NAC region results from hydrophobic aliphatic contacts, mostly between valine and alanine residues, and cation-π interactions between lysine and tyrosine. In addition, hydrogen bonds also seem to contribute to the compaction of the NAC region. The expansion of the C-terminal region is due to intraregional electrostatic repulsion and increased chain stiffness from several prolines. Overall, our study demonstrates the effectiveness of combining smFRET experiments with CG-MD simulations to investigate the key interactions in highly dynamic IDPs at the amino acid level.

Nonspecific interaction and overlap concentration influence macromolecular crowding effect on glucose oxidase activity

Macromolecular crowding can change kinetics of enzyme catalysis. How interaction between enzymes and neighboring macromolecules contributes to the crowding effect on enzyme catalysis has not been quantitatively revealed. In this study, crowding effects of dextran and poly(ethylene glycol) (PEG) on glucose oxidase (GOx) are studied. Fluorescence resonance energy transfer experiments show the high transfer efficiency and stable interaction between the dextran and GOx. Further fluorescence quenching analysis also proves that the association of the dextran-GOx pair can become stronger than that of the PEG-GOx pair. Dextrans with concentrations above or below their chain overlap concentrations (c*) reduce Michaelis constants (Km) of GOx catalysis by 90 % or 45 %, respectively, through volume exclusion mechanism, and in the meantime elevate the enzymatic efficiency (kcat/Km) by 8-fold or by 3-fold, respectively, which is more dramatic than that found in other enzymes before. Strong association between the enzyme and the dextran results in slow turnover rates (kcat). Intermediate crowding with weak to moderate affinity to the enzyme below the c* can tune the kcat higher than in the free state. Catalysis under crowded conditions is a joint effect of the enzyme-crowder nonspecific interaction, volume exclusion and overlap condition of the crowders.

Jinzhen Oral Liquid alleviates lipopolysaccharide-induced acute lung injury through modulating TLR4/MyD88/NF-κB pathway

BackgroundAcute lung injury (ALI) has the attribution of excessive inflammation of the lung. Jinzhen oral liquid (JO), a famous Chinese recipe used to treat ALI, has a favorable therapeutic effect on ALI. However, its anti-inflammatory mechanism has not been extensively studied. PurposeThis study was to elucidate the effects of JO on lipopolysaccharide (LPS)-induced ALI and its molecular mechanism. MethodsAn ALI model was established by intratracheal instillation of LPS (2 mg/50 μl). The open field experiment was carried out to explore the spontaneous movement and exploratory behavior of ALI mice. Cytokines levels concentrations (IL-6, IL-10 and TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA). Network pharmacology was used to predict the mechanism of JO against ALI. Immunofluorescence, co-immunoprecipitation, fluorescence resonance energy transfer (FRET), Western blot and RT-PCR were used to verify the molecular mechanisms of JO. ResultsThe in vivo results suggested that JO (1, 2, 4 g/kg) dose-dependently improved the exercise performance of mice and reduced the lung W/D weight ratio as well as the production of IL-6 and TNF-α, but increased the release of IL-10 in the ALI group. The network pharmacological analysis demonstrated that the Toll-like receptor (TLR) pathway might be the fundamental action mechanisms of JO against ALI. Immunofluorescence staining and co-immunoprecipitation analysis showed that JO decreased the expression levels of TLR4 and MyD88 and reduced their interaction in the lung tissue of ALI mice. Meanwhile, JO decreased nuclear translocation and phosphorylation of NF-κB P65. The results from cellular experiments were in line with those in vivo. The FRET experiment also confirmed that JO disturbed the interaction of TLR4 and MyD88. Subsequently, we also found that the six indicative components of JO have the similar therapeutic effect as JO. ConclusionsIn summary, we suggested that JO suppressed the TLR4/MyD88/NF-κB signaling pathway, thus inhibiting LPS-induced ALI in vitro and in vivo. The clarified mechanism provided an important theoretical basis and a novel treatment strategy for the ALI treatment of JO.

The mechanism of dwarf open reading frame (DWORF) activation of the sarcoplasmic reticulum calcium pump SERCA.

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitory transmembrane peptide phospholamban. A new transmembrane peptide regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. We have shown that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban (1). Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake, while phospholamban decreases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium, while phospholamban has a higher affinity for SERCA in the absence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium. Mutagenesis and structure-function analysis of DWORF reveals three important features required for SERCA activation: Pro15 promotes a helix-linker-helix structure of DWORF; the N-terminal amphipathic helix of DWORF perturbs the membrane bilayer; and the short transmembrane helix of DWORF contributes to hydrophobic mismatch and positions the amphipathic helix at the surface of the membrane bilayer. Our current understanding of the mechanism of SERCA activation by DWORF will be presented.

Integration of smFRET and molecular dynamics to characterize conformational dynamics of the spike protein of wild-type SARS-CoV-2 and its variants.

Since the rise of the COVID-19 pandemic, researchers have pushed for development of efficient therapeutics and vaccines to assist in preventing the severity of its viral effects. A major target has been the trimeric SARS-CoV-2 spike protein. There has been various experimental and computational efforts to characterize the structural dynamics of this protein at different stages of its mechanisms. Among these methods are single molecule fluorescence resonance energy transfer (smFRET) experiments as well as molecular dynamics (MD) simulations. The purpose of our current work here is to combine the existing data from smFRET experiments and MD simulations to provide a more complete picture of prefusion SARS-CoV-2 spike protein conformational dynamics. We have particularly examined the conformational dynamics of several variants of this protein in a comparative manner. Specifically, we have examined multiple computational methods based on “implicit screening” approach within different approximations to predict dye-dye distance and FRET efficiency distributions to provide reliable interpretations for both smFRET experimental data and MD simulation data of various spike protein variants including the wild-type, the alpha, beta, gamma, delta, and omega variants as well as the engineered spike protein used in the Moderna vaccine. This work sheds light on the structural heterogeneity of the spike protein in different variants of SARS-CoV-2.

Alzheimer's disease-related presenilin 1 M146L mutation disrupts SERCA2a regulation and increases propensity of Ca waves in ventricular cardiomyocytes.

Presenilin 1 (PS1) is a transmembrane protein expressed in the endoplasmic reticulum (ER) of different cell types. It has been shown that Ca mishandling due to PS1 mutations contributes to the Alzheimer's disease (AD)-related neurodegeneration. PS1 is also expressed in the heart. Several clinical studies revealed that PS1 mutations are associated with cardiomyopathies. We previously showed that PS1 interacts with the cardiac ER Ca ATPase (SERCA2a) and regulates its function. Here, we studied the role of AD-related PS1M146L mutation in regulation of intracellular Ca homeostasis. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1M146L revealed that the mutation does not disrupt the interaction between these two proteins. Measurements of SERCA2a-mediated Ca transport showed that at low ER Ca loads ([Ca]ER≤0.15 mM), both PS1WT and PS1M146L enhance ER Ca uptake by a similar level (∼40%) compared to control (no PS1 expression). At high ER Ca loads ([Ca]ER≥0.35 mM), however, PS1WT reduced ER Ca uptake and ER Ca load, whereas PS1M146L was less effective in regulating SERCA2a function. We used in vivo gene delivery in mouse cardiac wall to study the effect of PS1WT and PS1M146L overexpression on Ca regulation in ventricular myocytes. We found that both PS1WT and PS1M146L localized predominantly at the sarcoplasmic reticulum. Analysis of cytosolic Ca dynamics revealed that PS1WT overexpression in cardiomyocytes increases diastolic Ca and decreases Ca transient amplitudes. In contrast, PS1M146L expression increased propensity of spontaneous Ca waves. These results illustrate that the lack of SERCA2a regulation by PS1M146L at high ER Ca loads can lead to ER Ca overload and Ca waves. These defects in Ca regulation might contribute to the onset of cardiomyopathies in patients with PS1 mutations.

Negative allosteric modulation of the glucagon receptor by RAMP2.

Receptor activity-modifying proteins (RAMPs) modulate the activity of many Family B heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The glucagon receptor (GCGR), a Family B GPCR responsible for maintenance of proper blood sugar levels, interacts with RAMP2, though the purpose and consequence of this interaction is poorly understood. Using a series of biochemical and cell-based assays, we show that RAMP2 interacts with and broadly inhibits GCGR-induced downstream signaling. Hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) demonstrate that RAMP2 enhances local dynamics in select locations in and near the receptor extracellular domain (ECD) as well as at a key region in the 6th transmembrane helix, while single-molecule fluorescence resonance energy transfer (smFRET) experiments show that this RAMP2-induced ECD disorder results in inhibition of active and intermediate states of the intracellular face of the receptor. Using cryo-electron microscopy (cryoEM), we determined the structure of the GCGR-Gs complex at 2.9 Å resolution in the presence of RAMP2. RAMP2 apparently does not interact with GCGR in an ordered manner, yet the ECD of GCGR is indeed largely disordered in the presence of RAMP2. This disorder is accompanied by rearrangements of several key areas of the receptor, resulting in the formation of a likely unproductive complex. Together, our studies suggest that RAMP2 acts as a negative allosteric modulator of GCGR by enhancing conformational sampling of the ECD.

Chain length and amino acid composition dependence of coil-globule transition in intrinsically disordered proteins.

A fundamental question related to intrinsically disordered proteins (IDPs) is whether they undergo a coil-to-globule transition with changes in cosolvent conditions. IDPs are hetero-polymers and finite in size. The chain length and amino acid composition could lead to the varying extent of compaction in IDPs and the coil-to-globule transition (theta condition). To probe the theta condition in polypeptides, we studied the behavior of polyleucine and polyglutamine with six different chain lengths (N = 16 to 512) in various concentrations (0 to 8 M) of Urea (denaturant) and TMAO (osmolyte) using coarse-grained models and computer simulations. The Flory scaling exponent of polyleucine (polyglutamine) undergoes a transition from approximately 0.6 (0.6) to 0.3 (0.5) with increasing N in water. We observed that the theta condition for both the IDPs strongly depends on N and varies as N−0.5. Depending on N, polyglutamine (polyleucine) chains reach the theta condition at different [TMAO] ([TMAO] or [Urea]), confirming that in addition to chain length, the amino acid composition also influences the theta solvent condition for the IDPs. Moreover, the theta point for smaller chain lengths (N = 16 to 64) calculated using the structure factor mimicking the small-angle X-ray scattering (SAXS) experiments and using pair distances mimicking fluorescence resonance energy transfer (FRET) experiments are in disagreement. The theta condition computed using the structure factor and pair distances converge to the same point only at large N. As the theta condition is correlated with the critical condition of liquid-liquid phase separation (LLPS) for IDPs, we predict that LLPS propensity of IDPs will increase with N.

Time-resolved mechanisms of essential biological pathways.

The diversity of enzyme structure and function in living systems leads to exciting opportunities in biophysical research. Large numbers of these protein machines coordinate across space and time and undergo drastic changes in shape to carry out complex biological processes. My lab focuses on investigating precisely how genetic mutations impact the molecular function of key enzymes involved in DNA repair and/or RNA processing pathways. We use single molecule time-resolved fluorescence spectroscopy as a tool to explore in real time the protein-nucleic acid interactions and dynamic structural rearrangements that drive specific pathways. We are currently studying the detailed mechanisms SARS-CoV-2 viral RNA processing, ribosome assembly, and DNA mismatch repair. I will present recent results from pulsed interleaved excitation fluorescence resonance energy transfer (PIE-FRET) and fluorescence correlation spectroscopy (FCS) experiments. We aim to completely characterize the impact of mutations linked to disease on the molecular function of enzymes to understand how complex diseases develop at the molecular level and to identify new therapeutic targets.

Bioderived Lipoic Acid-Based Dynamic Covalent Nanonetworks of Poly(disulfide)s: Enhanced Encapsulation Stability and Cancer Cell-Selective Delivery of Drugs.

Dynamic covalent poly(disulfide)-based cross-linked nanoaggregates, termed nanonetworks (NNs), endowed with pH- and redox-responsive degradation features have been fabricated for stable noncovalent encapsulation and triggered cargo release in a controlled fashion. A bioderived lipoic acid-based Gemini surfactant-like amphiphilic molecule was synthesized for the preparation of nanoaggregates. It self-assembles by a entropy-driven self-assembly process in aqueous milieu. To further stabilize the self-assembled nanostructure, the core was cross-linked by ring-opening disulfide exchange polymerization (RODEP) of 1,2-dithiolane rings situated inside the core of the nanoaggregates. The cross-linked nanoaggregates, i.e., nanonetwork, are found to be stable in the presence of blood serum, and also, they maintain the self-assembled structure even below the critical aggregation concentration (CAC) as probed by dynamic light scattering (DLS) experiments. The nanonetwork showed almost 50% reduction in guest leakage compared to that of the nanoaggregates as shown by the release profile in the absence of stimuli, suggesting high encapsulation stability as evidenced by the fluorescence resonance energy transfer (FRET) experiment. The decross-linking of the nanonetwork occurs in response to redox and pH stimuli due to disulfide reduction and β-thioester hydrolysis, respectively, thus empowering disassembly-mediated controlled cargo release up to ∼87% for 55 h of incubation. The biological evaluation of the doxorubicin (DOX)-loaded nanonetwork revealed environment-specific surface charge modulation-mediated cancer cell-selective cellular uptake and cytotoxicity. The benign nature of the nanonetwork toward normal cells makes the system very promising in targeted drug delivery applications. Thus, the ease of synthesis, nanonetwork fabrication reproducibility, robust stability, triggered drug release in a controlled fashion, and cell-selective cytotoxicity behavior, we believe, will make the system a potential candidate in the development of robust materials for chemotherapeutic applications.

Characterization of the Binding Properties of Sorafenib to c-MYC G-Quadruplexes: Evidence for Screening Potential Ligands.

Sorafenib (Sor) is a multitarget kinase inhibitor used clinically to treat hepatocellular carcinoma and renal cancer. In this study, the interaction mechanism of Sor with c-MYC G-quadruplexes (G4) was investigated at the molecular level by computer-aided means and experiments. Molecular docking results predicted the binding of Sor to the groove of G4. Molecular dynamics (MD) simulations were used to evaluate the effect of ligand binding to G4. Ultraviolet (UV), fluorescence spectroscopy, and viscosity experiments showed that the binding site was in the groove. The UV and fluorescence titration results showed that compared with traditional G4 ligands represented by compound meso-tetra (N-methyl-4-pyridyl) porphine (TmPyP4), Sor has a lower affinity for G4. Likewise, results from fluorescence resonance energy transfer (FRET) experiments suggested that Sor could have a limited ability to stabilize G4, but it was not as prominent as that of TmPyP4. Time-resolved fluorescence spectroscopy again supported the results from steady-state fluorescence spectroscopy, indicating that a static quenching mechanism mainly drove the process. Studying the interaction mechanism of Sor and c-MYC may inspire the screening of new, selective c-MYC G4 ligands and provide ideas for the design of drugs with good stability, low toxicity, and specific targeting of G4.

Tumor acidity-induced surface charge modulation in covalent nanonetworks for activated cellular uptake: targeted delivery of anticancer drugs and selective cancer cell death.

A β-thioester and tertiary amine based covalently cross-linked nanoassembly coined as a nanonetwork (NN) endowed with dual pH responsive features (tumor acidity induced surface charge modulation and endosomal pH triggered controlled degradation) has been designed and synthesized for stable sequestration and sustained release of drug molecules in response to endosomal pH. An amphiphile integrated with tertiary amine and acrylate (ATA) functionalities was synthesized to fabricate the nanonetwork. This amphiphile showed entropically driven self-assembly and micellar nanostructures (nanoassemblies), which can sequester hydrophobic drug molecules at neutral pH. To further stabilize the nanoassemblies and the sequestered drug molecules even below its critical aggregation concentration (CAC), the micellar core was cross-linked via the thiol-acrylate Michael addition click reaction to generate multiple copies of acid labile β-thioester functionalities in the core, which undergo slow hydrolysis at endosomal pH (∼5.0), thus enabling sustained release of the anti-cancer drug doxorubicin at endosomal pH. The nanonetworks showed a significant decrease in drug leakage compared to the nanoassemblies (NAs), which was also justified by a low leakage coefficient calculated from the fluorescence resonance energy transfer experiment. The NN also exhibited dilution insensitivity and high serum stability, whereas the NA disassembled upon dilution and during serum treatment. The biological evaluation revealed tumor extracellular matrix pH (∼6.4-6.8) induced surface charge modulation and cancer cell (HeLa) selective activated cellular uptake of the doxorubicin loaded nanonetwork (NN-DOX). In contrast, the benign nature of NN-DOX towards normal cells (H9c2) suggests excellent cell specificity. Thus, we believe that the ease of synthesis, nanonetwork fabrication reproducibility, robust stability, smart nature of tumor microenvironment sensitive surface charge modulation, boosted tumoral-cell uptake, and triggered drug release will make this system a potential nanomedicine for chemotherapeutic treatments.

Learning continuous potentials from smFRET

Potential energy landscapes are useful models in describing events such as protein folding and binding. While single-molecule fluorescence resonance energy transfer (smFRET) experiments encode information on continuous potentials for the system probed, including rarely visited barriers between putative potential minima, this information is rarely decoded from the data. This is because existing analysis methods often model smFRET output assuming, from the onset, that the system probed evolves in a discretized state space to be analyzed within a hidden Markov model (HMM) paradigm. By contrast, here, we infer continuous potentials from smFRET data without discretely approximating the state space. We do so by operating within a Bayesian nonparametric paradigm by placing priors on the family of all possible potential curves. As our inference accounts for a number of required experimental features raising computational cost (such as incorporating discrete photon shot noise), the framework leverages a structured-kernel-interpolation Gaussian process prior to help curtail computational cost. We show that our structured-kernel-interpolation priors for potential energy reconstruction from smFRET analysis accurately infers the potential energy landscape from a smFRET binding experiment. We then illustrate advantages of structured-kernel-interpolation priors for potential energy reconstruction from smFRET over standard HMM approaches by providing information, such as barrier heights and friction coefficients, that is otherwise inaccessible to HMMs.

Simultaneous crosslinking of CD20 and CD38 receptors by drug-free macromolecular therapeutics enhances B cell apoptosis in vitro and in vivo.

Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without the need of low molecular weight drugs. Programmed cell death is initiated via a biomimetic receptor crosslinking strategy using a two-step approach: i) recognition of cell surface antigen by a morpholino oligonucleotide-modified antibody Fab' fragment (Fab'-MORF1), ii) followed by crosslinking with a multivalent effector motif - human serum albumin (HSA) grafted with multiple complementary morpholino oligonucleotides (HSA-(MORF2)x). This approach is effective in vitro, in vivo, and ex vivo on cells from patients diagnosed with various B cell malignancies. We have previously demonstrated DFMT can be applied to crosslink CD20 and CD38 receptors to successfully initiate apoptosis. Herein, we show simultaneous engagement, and subsequent crosslinking of both targets ("heteroreceptor crosslinking"), can further enhance the apoptosis induction capacity of this system. To accomplish this, we incubated Raji (CD20+; CD38+) cells simultaneously with anti-CD20 and anti-CD38 Fab'-MORF1 conjugates, followed by addition of the macromolecular crosslinker, HSA-(MORF2)x to co-cluster the bound receptors. Fab' fragments from Rituximab and Obinutuzumab were employed in the synthesis of anti-CD20 bispecific engagers (Fab'RTX-MORF1 and Fab'OBN-MORF1), whereas Fab' fragments from Daratumumab and Isatuximab (Fab'DARA-MORF1 and Fab'ISA-MORF1) targeted CD38. All heteroreceptor crosslinking DFMT combinations demonstrated potent apoptosis induction and exhibited synergistic effects as determined by Chou-Talalay combination index studies (CI<1). In vitro fluorescence resonance energy transfer (FRET) experiments confirmed the co-clustering of the two receptors on the cell surface in response to the combination treatment. The source of this synergistic therapeutic effect was further explored by evaluating the effect of combination DFMT on key apoptosis signaling events such as mitochondrial depolarization, caspase activation, lysosomal enlargement, and homotypic cell adhesion. Finally, a xenograft mouse model of CD20+/CD38+ Non Hodgkin lymphoma was employed to demonstrate in vivo the enhanced efficacy of the heteroreceptor-crosslinking DFMT design versus single-target systems.