Unraveling the molecular dynamics of sugammadex-rocuronium complexation: A blueprint for cyclodextrin drug design.

Sugammadex, marketed as Bridion™, is an approved cyclodextrin (CD) based drug for the reversal of neuromuscular blockade in adults undergoing surgery. Sugammadex forms an inclusion complex with the neuromuscular blocking agent (NMBA) rocuronium, allowing rapid reversal of muscle paralysis. In silico methods have been developed for studying CD inclusion complexes, aimed at accurately predicting their structural, energetic, dynamic, and kinetic properties, as well as binding constants. Here, a computational study aimed at characterizing the sugammadex-rocuronium system from the perspective of docking calculations, free molecular dynamics (MD) simulations, and biased metadynamics simulations with potential of mean force (PMF) calculations is presented. The aim is to provide detailed information about this system, as well as to use it as a model system for validation of the methods. This method predicts results in line with experimental evidence for both the optimal structure and the quantitative value for the binding constant. Interestingly, there is a less profound preference for the orientation than might be assumed based on electrostatic interactions, suggesting that both orientations may exist in solution. These results show that this technology can efficiently analyze CD inclusion complexes and could be used to facilitate the development and optimization of novel applications for CDs.

Cyclodextrins: Establishing building blocks for AI-driven drug design by determining affinity constants in silico.

Cyclodextrins (CDs) are cyclic carbohydrate polymers that hold significant promise for drug delivery and industrial applications. Their effectiveness depends on their ability to encapsulate target molecules with strong affinity and specificity, but quantifying affinities in these systems accurately is challenging for a variety of reasons. Computational methods represent an exceptional complement to in vitro assays because they can be employed for existing and hypothetical molecules, providing high resolution structures in addition to a mechanistic, dynamic, kinetic, and thermodynamic characterization. Here, we employ potential of mean force (PMF) calculations obtained from guided metadynamics simulations to characterize the 1:1 inclusion complexes between four different modified ßCDs, with different type, number, and location of substitutions, and two sterol molecules (cholesterol and 7-ketocholesterol). Our methods, validated for reproducibility through four independent repeated simulations per system and different post processing techniques, offer new insights into the formation and stability of CD-sterol inclusion complexes. A systematic distinct orientation preference where the sterol tail projects from the CD's larger face and significant impacts of CD substitutions on binding are observed. Notably, sampling only the CD cavity's wide face during simulations yielded comparable binding energies to full-cavity sampling, but in less time and with reduced statistical uncertainty, suggesting a more efficient approach. Bridging computational methods with complex molecular interactions, our research enables predictive CD designs for diverse applications. Moreover, the high reproducibility, sensitivity, and cost-effectiveness of the studied methods pave the way for extensive studies of massive CD-ligand combinations, enabling AI algorithm training and automated molecular design.

Selective Removal of 7KC by a Novel Atherosclerosis Therapeutic Candidate Reverts Foam Cells to a Macrophage-like Phenotype.

The removal of the toxic oxidized cholesterol, 7-ketocholesterol (7KC), from cells through the administration of therapeutics has the potential to treat atherosclerosis and various other pathologies. While cholesterol is a necessary building block for homeostasis, oxidation of cholesterol can lead to the formation of toxic oxysterols involved in various pathologies, the most prominent of which is 7KC, which is formed through the non-enzymatic oxidation of cholesterol. Oxidized LDL (oxLDL) particles, highly implicated in heart disease, contain high levels of 7KC, and molecular 7KC is implicated in the pathogenesis of numerous diseases, including multiple sclerosis, hypercholesterolemia, sickle cell anemia, and multiple age related diseases. Of particular interest is the role of 7KC in the progression of atherosclerosis, with several studies associating elevated levels of 7KC with the etiology of the disease or in the transition of macrophages to foam cells. This research aims to elucidate the molecular mechanisms of UDP-003, a novel therapeutic, in mitigating the harmful effects of 7KC in mouse and human monocyte and macrophage cell lines. Experimental evidence demonstrates that administration of UDP-003 can reverse the foam cell phenotype, rejuvenating these cells by returning phagocytic function and decreasing both reactive oxygen species (ROS) and intracellular lipid droplet accumulation. Furthermore, our data suggests that the targeted removal of 7KC from foam cells with UDP-003 can potentially prevent and reverse atherosclerotic plaque formation. UDP-003 has the potential to be the first disease-modifying therapeutic approach to treating atherosclerotic disease.

The Mitochondrial Genome in Aging and Disease and the Future of Mitochondrial Therapeutics.

Mitochondria are intracellular organelles that utilize nutrients to generate energy in the form of ATP by oxidative phosphorylation. Mitochondrial DNA (mtDNA) in humans is a 16,569 base pair double-stranded circular DNA that encodes for 13 vital proteins of the electron transport chain. Our understanding of the mitochondrial genome's transcription, translation, and maintenance is still emerging, and human pathologies caused by mtDNA dysfunction are widely observed. Additionally, a correlation between declining mitochondrial DNA quality and copy number with organelle dysfunction in aging is well-documented in the literature. Despite tremendous advancements in nuclear gene-editing technologies and their value in translational avenues, our ability to edit mitochondrial DNA is still limited. In this review, we discuss the current therapeutic landscape in addressing the various pathologies that result from mtDNA mutations. We further evaluate existing gene therapy efforts, particularly allotopic expression and its potential to become an indispensable tool for restoring mitochondrial health in disease and aging.

Rapid enrichment of mitochondria from mammalian cell cultures using digitonin.

We describe here a simple method to enrich mitochondrial fractions from mammalian cells for downstream analyses in the lab. Mitochondria purification involves cell lysis followed by separation of the organelles from the rest of the cellular components. Here, we use detergent to rupture the cell membrane of mammalian cells followed by differential centrifugation to enrich the organelles. Optimum conditions with respect to detergent concentration, time, sample size, and yield are discussed. The method's utility in downstream analyses and ease of processing multiple samples simultaneously is also described. All the reagents in this method can be assembled in-house, are economical, and are comparable, if not superior, to commercially available kits in terms of mitochondrial yield and integrity. • Rapid enrichment of mitochondria from mammalian cells using commonly available reagents. • Multiple samples can be processed simultaneously. • Works over a wide range of sample size (1 million to 100 million cells).

Cyclodextrin dimers: A versatile approach to optimizing encapsulation and their application to therapeutic extraction of toxic oxysterols.

We have developed a novel class of specifically engineered, dimerized cyclodextrin (CD) nanostructures for the encapsulation of toxic biomolecules such as 7-ketocholesterol (7KC). 7KC accumulates over time and causes dysfunction in many cell types, linking it to several age-related diseases including atherosclerosis and age-related macular degeneration (AMD). Presently, treatments for these diseases are invasive, expensive, and show limited benefits. CDs are cyclic glucose oligomers utilized to capture small, hydrophobic molecules. Here, a combination of in silico, in vitro, and ex vivo methods is used to implement a synergistic rational drug design strategy for developing CDs to remove atherogenic 7KC from cells and tissues. Mechanisms by which CDs encapsulate sterols are discussed, and we conclude that covalently linked head-to-head dimers of ßCDs have substantially improved affinity for 7KC compared to monomers. We find that inclusion complexes can be stabilized or destabilized in ways that allow the design of CD dimers with increased 7KC selectivity while maintaining an excellent safety profile. These CD dimers are being developed as therapeutics to treat atherosclerosis and other debilitating diseases of aging.

Codon optimization is an essential parameter for the efficient allotopic expression of mtDNA genes.

Mutations in mitochondrial DNA can be inherited or occur de novo leading to several debilitating myopathies with no curative option and few or no effective treatments. Allotopic expression of recoded mitochondrial genes from the nucleus has potential as a gene therapy strategy for such conditions, however progress in this field has been hampered by technical challenges. Here we employed codon optimization as a tool to re-engineer the protein-coding genes of the human mitochondrial genome for robust, efficient expression from the nucleus. All 13 codon-optimized constructs exhibited substantially higher protein expression than minimally-recoded genes when expressed transiently, and steady-state mRNA levels for optimized gene constructs were 5-180 fold enriched over recoded versions in stably-selected wildtype cells. Eight of thirteen mitochondria-encoded oxidative phosphorylation (OxPhos) proteins maintained protein expression following stable selection, with mitochondrial localization of expression products. We also assessed the utility of this strategy in rescuing mitochondrial disease cell models and found the rescue capacity of allotopic expression constructs to be gene specific. Allotopic expression of codon optimized ATP8 in disease models could restore protein levels and respiratory function, however, rescue of the pathogenic phenotype for another gene, ND1 was only partially successful. These results imply that though codon-optimization alone is not sufficient for functional allotopic expression of most mitochondrial genes, it is an essential consideration in their design.

7-Ketocholesterol in disease and aging.

7-Ketocholesterol (7KC) is a toxic oxysterol that is associated with many diseases and disabilities of aging, as well as several orphan diseases. 7KC is the most common product of a reaction between cholesterol and oxygen radicals and is the most concentrated oxysterol found in the blood and arterial plaques of coronary artery disease patients as well as various other disease tissues and cell types. Unlike cholesterol, 7KC consistently shows cytotoxicity to cells and its physiological function in humans or other complex organisms is unknown. Oxysterols, particularly 7KC, have also been shown to diffuse through membranes where they affect receptor and enzymatic function. Here, we will explore the known and proposed mechanisms of pathologies that are associated with 7KC, as well speculate about the future of 7KC as a diagnostic and therapeutic target in medicine.

Stable nuclear expression of ATP8 and ATP6 genes rescues a mtDNA Complex V null mutant.

We explore the possibility of re-engineering mitochondrial genes and expressing them from the nucleus as an approach to rescue defects arising from mitochondrial DNA mutations. We have used a patient cybrid cell line with a single point mutation in the overlap region of the ATP8 and ATP6 genes of the human mitochondrial genome. These cells are null for the ATP8 protein, have significantly lowered ATP6 protein levels and no Complex V function. Nuclear expression of only the ATP8 gene with the ATP5G1 mitochondrial targeting sequence appended restored viability on Krebs cycle substrates and ATP synthesis capabilities but, failed to restore ATP hydrolysis and was insensitive to various inhibitors of oxidative phosphorylation. Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6 Thus, we report the stable allotopic expression, import and function of two mitochondria encoded genes, ATP8 and ATP6, resulting in simultaneous rescue of the loss of both mitochondrial proteins.

Differentiation rather than aging of muscle stem cells abolishes their telomerase activity.

A general feature of stem cells is the ability to routinely proliferate to build, maintain, and repair organ systems. Accordingly, embryonic and germline, as well as some adult stem cells, produce the telomerase enzyme at various levels of expression. Our results show that, while muscle is a largely postmitotic tissue, the muscle stem cells (satellite cells) that maintain this biological system throughout adult life do indeed display robust telomerase activity. Conversely, primary myoblasts (the immediate progeny of satellite cells) quickly and dramatically downregulate telomerase activity. This work thus suggests that satellite cells, and early transient myoblasts, may be more promising therapeutic candidates for regenerative medicine than traditionally utilized myoblast cultures. Muscle atrophy accompanies human aging, and satellite cells endogenous to aged muscle can be triggered to regenerate old tissue by exogenous molecular cues. Therefore, we also examined whether these aged muscle stem cells would produce tissue that is "young" with respect to telomere maintenance. Interestingly, this work shows that the telomerase activity in muscle stem cells is largely retained into old age wintin inbred "long" telomere mice and in wild-derived short telomere mouse strains, and that age-specific telomere shortening is undetectable in the old differentiated muscle fibers of either strain. Summarily, this work establishes that young and old muscle stem cells, but not necessarily their progeny, myoblasts, are likely to produce tissue with normal telomere maintenance when used in molecular and regenerative medicine approaches for tissue repair.

Notch signaling pathway and tissue engineering.

The Notch pathway is a signaling network essential for proper organ development in an embryo, and is indispensable for tissue regeneration in the adult. This key regulatory signaling network is evolutionarily conserved in all metazoans and is continually utilized for the building, maintenance and repair of diverse organs and tissues. Importantly, dysfunctions in the Notch pathway have been demonstrated to result in oncogenic transformation, such as in lymphoid cancers, and have been linked to the pathogenesis of several inherited human diseases. Therefore, the ability to regulate Notch signaling intensity both positively and negatively has a very high therapeutic relevance. Adapting this pathway for tissue engineering applications has great potential to spear-head the development of smart biomaterials to deliberately control cell-fate decisions and lead to designer ex vivo morphogenesis. This review describes the components of Notch-specific signal transduction, presents the role of the Notch signaling network in constructing and repairing multiple organ systems, summarizes the Notch-related pathologies, outlines current advances in the deliberate modulation of the Notch pathway in bioengineering applications, and introduces future perspectives on the use of Notch pathway manipulations as a powerful universal tool in tissue engineering and in the orchestration of stem cell responses. This review also summarizes the existing bioengineering methods most suitable for the deliberate manipulation of Notch signaling, such as smart biomaterials able to pattern Notch ligands or to create gradients of Notch agonists and antagonists. Such methods will likely facilitate the engineering and dynamic remodeling of tissues composed of stem, progenitor and differentiated cells derived from an initially equivalent cell population.

TPP1 is a homologue of ciliate TEBP-beta and interacts with POT1 to recruit telomerase.

Telomere dysfunction may result in chromosomal abnormalities, DNA damage responses, and even cancer. Early studies in lower organisms have helped to establish the crucial role of telomerase and telomeric proteins in maintaining telomere length and protecting telomere ends. In Oxytricha nova, telomere G-overhangs are protected by the TEBP-alpha/beta heterodimer. Human telomeres contain duplex telomeric repeats with 3' single-stranded G-overhangs, and may fold into a t-loop structure that helps to shield them from being recognized as DNA breaks. Additionally, the TEBP-alpha homologue, POT1, which binds telomeric single-stranded DNA (ssDNA), associates with multiple telomeric proteins (for example, TPP1, TIN2, TRF1, TRF2 and RAP1) to form the six-protein telosome/shelterin and other subcomplexes. These telomeric protein complexes in turn interact with diverse pathways to form the telomere interactome for telomere maintenance. However, the mechanisms by which the POT1-containing telosome communicates with telomerase to regulate telomeres remain to be elucidated. Here we demonstrate that TPP1 is a putative mammalian homologue of TEBP-beta and contains a predicted amino-terminal oligonucleotide/oligosaccharide binding (OB) fold. TPP1-POT1 association enhanced POT1 affinity for telomeric ssDNA. In addition, the TPP1 OB fold, as well as POT1-TPP1 binding, seemed critical for POT1-mediated telomere-length control and telomere-end protection in human cells. Disruption of POT1-TPP1 interaction by dominant negative TPP1 expression or RNA interference (RNAi) resulted in telomere-length alteration and DNA damage responses. Furthermore, we offer evidence that TPP1 associates with the telomerase in a TPP1-OB-fold-dependent manner, providing a physical link between telomerase and the telosome/shelterin complex. Our findings highlight the critical role of TPP1 in telomere maintenance, and support a yin-yang model in which TPP1 and POT1 function as a unit to protect human telomeres, by both positively and negatively regulating telomerase access to telomere DNA.

A critical role for TPP1 and TIN2 interaction in high-order telomeric complex assembly.

Mammalian telomeric proteins function through dynamic interactions with each other and telomere DNA. We previously reported the formation of a high-molecular-mass telomeric complex (the mammalian telosome) that contains the six core proteins TRF1, TRF2, RAP1, TIN2, POT1, and TPP1 (formerly named PTOP/PIP1/TINT1) and mediates telomere end-capping and length control. In this report, we sought to elucidate the mechanism of six-protein complex (or shelterin) formation and the function of this complex. Through reconstitution experiments, we demonstrate here that TIN2 and TPP1 are key components in mediating the six-protein complex assembly. We demonstrate that not only TIN2 but also TPP1 are required to bridge the TRF1 and TRF2 subcomplexes. Specifically, TPP1 helps to stabilize the TRF1-TIN2-TRF2 interaction and promote six-protein complex formation. Consistent with this model, overexpression of TPP1 enhanced TIN2-TRF2 association. Conversely, knocking down TPP1 reduced the ability of endogenous TRF1 to associate with the TRF2 complex. Our results suggest that coordinated interactions among TPP1, TIN2, TRF1, and TRF2 may ensure robust assembly of the telosome, telomere targeting of its subunits, and, ultimately, regulated telomere maintenance.

Telosome, a mammalian telomere-associated complex formed by multiple telomeric proteins.

In mammalian cells, telomere-binding proteins TRF1 and TRF2 play crucial roles in telomere biology. They interact with several other telomere regulators including TIN2, PTOP, POT1, and RAP1 to ensure proper maintenance of telomeres. TRF1 and TRF2 are believed to exert distinct functions. TRF1 forms a complex with TIN2, PTOP, and POT1 and regulates telomere length, whereas TRF2 mediates t-loop formation and end protection. However, whether cross-talk occurs between the TRF1 and TRF2 complexes and how the signals from these complexes are integrated for telomere maintenance remain to be elucidated. Through gel filtration and co-immunoprecipitation experiments, we found that TRF1 and TRF2 are in fact subunits of a telomere-associated high molecular weight complex (telosome) that also contains POT1, PTOP, RAP1, and TIN2. We demonstrated that the TRF1-interacting protein TIN2 binds TRF2 directly and in vivo, thereby bridging TRF2 to TRF1. Consistent with this multi-protein telosome model, stripping TRF1 off the telomeres by expressing tankyrase reduced telomere recruitment of not only TIN2 but also TRF2. These results help to unify previous observations and suggest that telomere maintenance depends on the multi-subunit telosome.

PTOP interacts with POT1 and regulates its localization to telomeres.

Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1-12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA). In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP-POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.

The human Rap1 protein complex and modulation of telomere length.

Proper maintenance of telomere length and structure is necessary for normal proliferation of mammalian cells. Mammalian telomere length is regulated by a number of proteins including human repressor activator protein (hRap1), a known association factor of TRF2. To further delineate hRap1 function and its associated proteins, we affinity-purified and identified the hRap1 protein complex through mass spectrometry analysis. In addition to TRF2, we found DNA repair proteins Rad50, Mre11, PARP1 (poly(ADP-ribose) polymerase), and Ku86/Ku70 to be in this telomeric complex. We demonstrated by deletional analysis that Rad-50/Mre-11 and Ku86 were recruited to hRap1 independent of TRF2. PARP1, however, most likely interacted with hRap1 through TRF2. Interestingly, knockdown of endogenous hRap1 expression by small hairpin interference RNA resulted in longer telomeres. In addition, overexpression of full-length and mutant hRap1 that lacked the BRCA1 C-terminal domain functioned as dominant negatives and extended telomeres. Deletion of a novel linker domain of hRap1 (residues 199-223), however, abolished the dominant negative effect of hRap1 overexpression. These results indicate that hRap1 negatively regulates telomere length in vivo and suggest that the linker region of hRap1 may modulate the recruitment of negative regulators of telomere length.

Using the stepladder technique to facilitate the performance of audioconferencing groups.

Organizational workforces are becoming increasingly dispersed. To facilitate communications among individuals or groups of people located in a number of different locations, teleconferencing technologies, such as audioconferencing, have been developed. The authors examined whether a structural group intervention, the stepladder technique, can facilitate the task performance of 4-person groups (n = 52) when using audioconferencing. Consistent with research conducted on face-to-face groups, the stepladder technique was found to facilitate the decision-making performance of groups interacting via audioconference. The authors postulated that certain structural elements of the stepladder technique compensate for obstacles inherent in nonvisual communications. Supplementary analyses examined best member influence and the existence of order of entry effects into the stepladder process.