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Melatonin MT 1 and MT 2 receptors represent attractive drug targets for the treatment of various disorders. However, the high conservation of the melatonin binding pocket has hindered the development of subtype-selective compounds. By leveraging on the recently resolved crystal structures of MT 1 and MT 2 receptors, this study aims to elucidate the structural basis of MT 2 -selectivity of a panel of isoquinolinone derivatives. Molecular modelling and ligand docking approaches were employed to predict residues involved in forming interactions with the MT 2 -selective isoquinolinones. Seven conserved residues (Asn175, His208, Trp264, Asn268, Gly271, Tyr294 and Tyr298) were selected as targets for site-directed mutagenesis. Ca2+ mobilization, cAMP inhibition, phosphorylation of extracellular signal-regulated kinase, and ligand binding assays were performed to functionally characterize the receptor mutants in transfected CHO cells. Unlike melatonin, isoquinolinones bearing a 3-methoxybenzyloxyl substituent were unaffected by alanine substitution at His208 of MT 2. Although alanine substitutions at Tyr294 or Tyr298 reduced the potency of melatonin and some isoquinolinones on MT 2 , similar mutations on MT 1 allowed five hitherto ineffective isoquinolinones to act as agonists. An isoquinolinone antagonist bearing a 4-methoxybenzyloxyl moiety turned into an agonist at MT 2 mutants with alanine substitutions at His208, Tyr294 or Tyr298. A subset of residues is apparently involved in forming a hydrophobic binding cavity to confer selectivity upon the aromatic substituent of isoquinolinone compounds. Two conserved tyrosine residues on transmembrane helix 7 may confer ligand selectivity at MT 1 and MT 2 receptors, while a conserved histidine on transmembrane helix 5 is apparently involved in receptor activation. [ABSTRACT FROM AUTHOR]

Serine-based β-lactamases of Class A, C and D all rely on a key water molecule to hydrolyze and inactivate β-lactam antibiotics. This process involves two conserved catalytic steps. In the first acylation step, the β-lactam antibiotic forms an acyl-enzyme intermediate (ES*) with the catalytic serine residue. In the second deacylation step, an activated water molecule serves as nucleophile (WAT_Nu) to attack ES* and release the inactivated β-lactam. The coordination and activation of WAT_Nu is not fully understood. Using time-resolved x-ray crystallography and QM/MM simulations, we analyzed three intermediate structures of Class A β-lactamase PenP as it slowly hydrolyzed cephaloridine. WAT_Nu is centrally located in the apo structure but becomes slightly displaced away by ES* in the post-acylation structure. In the deacylation structure, WAT_Nu moves back and is positioned along the Bürgi–Dunitz trajectory with favorable energetic profile to attack ES*. Unexpectedly, WAT_Nu is also found to adopt a catalytically incompetent conformation in the deacylation structure forming a hydrogen bond with ES*. Our results reveal that ES* plays a significant role in coordinating and activating WAT_Nu through subtle yet distinct interactions at different stages of the catalytic process. These interactions may serve as potential targets to circumvent β-lactamase-mediated antibiotic resistance. [ABSTRACT FROM AUTHOR]

RNA polymerase transcribes certain genomic loci with higher errors rates. These transcription error-enriched genomic loci (TEELs) have implications in disease. Current deep-sequencing methods cannot distinguish TEELs from post-transcriptional modifications, stochastic transcription errors, and technical noise, impeding efforts to elucidate the mechanisms linking TEELs to disease. Here, we describe background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) to discern genomic regions enriched for transcription misincorporations. EmPC-seq innovatively combines a nuclear run-on assay for capturing nascent RNA before post-transcriptional modifications, a circular-sequencing step that sequences the same nascent RNA molecules multiple times to improve accuracy, and a statistical model for distinguishing error-enriched regions among stochastic polymerase errors. Applying EmPC-seq to the ribosomal RNA transcriptome, we show that TEELs of RNA polymerase I are not randomly distributed but clustered together, with higher error frequencies at nascent transcript 3′ ends. Our study establishes a reliable method of identifying TEELs with nucleotide precision, which can help elucidate their molecular origins. Unlabelled Image • EmPC-seq combines experimental, bioinformatics, and statistical methods to identify a high confidence set of transcriptional mutation-prone positions within genes, eliminating typically confounding factors arising from coverage biases, sequencing noise, alignment artifacts, and background mutations, • EmPC-seq affirmatively shows that transcriptional mutation-prone regions cluster together. • EmPC-seq allows single transcript analyses to investigate of the molecular origin of transcriptional errors, and we show that transcriptional mutations are enriched at the active site. • EmPC-seq can be widely applicable for the study of transcription mutations in various model systems including virus, bacteria, and mammalian cells. [ABSTRACT FROM AUTHOR]

In a typical biomolecular simulation using Atomic Multipole Optimized Energetics for Biomolecular Applications (AMOEBA) force field, the vast majority molecules in the simulation box consist of water, and these water molecules consume the most CPU power due to the explicit mutual induction effect. To improve the computational efficiency, we here develop two new nonpolarizable water models (with flexible bonds and fixed charges) that are compatible with AMOEBA solute: the 3‐site AW3C and 5‐site AW5C. To derive the force‐field parameters for AW3C and AW5C, we fit to six experimental liquid thermodynamic properties: liquid density, enthalpy of vaporization, dielectric constant, isobaric heat capacity, isothermal compressibility and thermal expansion coefficient, at a broad range of temperatures from 261.15 to 353.15 K under 1.0 atm pressure. We further validate our AW3C and AW5C water models by showing that they can well reproduce the radial distribution function g(r), self‐diffusion constant D, and hydration free energy from the AMOEBA03 water model and the experimental observations. Furthermore, we show that our AW3C and AW5C water models can greatly accelerate (>5 times) the bulk water as well as biomolecular simulations when compared to AMOEBA water. Specifically, we demonstrate that the applications of AW3C and AW5C water models to simulate a DNA duplex lead to a threefold acceleration, and in the meanwhile well maintain the structural properties as the fully polarizable AMOEBA water. We expect that our AW3C and AW5C water models hold great promise to be widely applied to simulate complex bio‐molecules using the AMOEBA force field. [ABSTRACT FROM AUTHOR]

• We propose a joint training framework where the knowledge of ANN is consistently transferred to the SNN during training. • We factorize the parameters of both ANN and SNN and restrict them to have the same singular vectors while learning different singular values. • Extensive experiments are conducted on vision benchmarks, demonstrating that our method reaches SOTA. Emerged as a biology-inspired method, Spiking Neural Networks (SNNs) mimic the spiking nature of brain neurons and have received lots of research attention. SNNs deal with binary spikes as their activation and therefore derive extreme energy efficiency on hardware. However, it also leads to an intrinsic obstacle that training SNNs from scratch requires a re-definition of the firing function for computing gradient. Artificial Neural Networks (ANNs), however, are fully differentiable to be trained with gradient descent. In this paper, we propose a joint training framework of ANN and SNN, in which the ANN can guide the SNN's optimization. This joint framework contains two parts: First, the knowledge inside ANN is distilled to SNN by using multiple branches from the networks. Second, we restrict the parameters of ANN and SNN, where they share partial parameters and learn different singular weights. Extensive experiments over several widely used network structures show that our method consistently outperforms many other state-of-the-art training methods. For example, on the CIFAR100 classification task, the spiking ResNet-18 model trained by our method can reach to 77.39 % top-1 accuracy with only 4 time steps. [ABSTRACT FROM AUTHOR]

• ATRA decreased Hepa1-6 tumor burden and tumor angiogenesis and fibrosis. • ATRA decreased the population of MDSCs. • ATRA decreased H22 tumor burden and tumor angiogenesis and fibrosis. • ATRA decreased protumor myeloid immune subsets. • ATRA increased cytotoxic T cell infiltration. Hepatocellular carcinoma is characterized by a high infiltration of myeloid-derived suppressor cells (MDSC), which are key drivers of maintaining the immunosuppressive tumor microenvironment. Therefore, targeting MDSCs will improve immunotherapies for cancers. It has been shown that all-trans retinoic acid (ATRA) can differentiate MDSCs into mature myeloid cells. However, whether ATRA suppression of MDSCs function could inhibit the growth of liver cancer remains unknown. Here we found that ATRA significantly inhibited hepatocellular carcinoma promotion, tumor cell proliferation, and angiogenesis markers. Moreover, ATRA decreased the number of mononuclear myeloid-derived suppressor cells (M−MDSCs), granulocytic myeloid-derived suppressor cells (G-MDSCs) and tumor-associated macrophages (TAMs) in spleens. In addition, ATRA significantly reduced the intratumoral infiltrating G-MDSCs and the expression of protumor immunosuppressive molecules (arginase 1, iNOS, IDO and S100A8 + A9), which was accompanied by increased cytotoxic T cell infiltration. Our study demonstrates that ATRA not only has direct intrinsic inhibitory effect on tumor angiogenesis and fibrosis, but also reeducates the tumor microenvironment toward an antitumor phenotype by altering the relative proportion between protumor and antitumor immune cells. This information introduces ATRA as a potential druggable target for treatment of hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Abstract M2-polarized tumor-associated macrophages (M2-TAMs) infiltrating the tumor microenvironment contribute to hepatocellular carcinoma (HCC) progression. It was reported that cancer cells undergoing EMT will acquire stemness characteristics. Here, the HCC SMMC-7721 cell line was co-cultured with M2-TAMs polarized from THP-1 cells in vitro. In in vivo studies, we used nude mice subcutaneous tumor model to test whether the growth of the tumor was affected by M2-TAMs. Subsequently, EMT, stemness and Wnt/β-catenin pathway related markers were detected in cells and subcutaneous tumor tissues. TNF-α was also assessed in both the co-culture system supernatants and in nude mice serum. We found that SMMC-7721 underwent EMT and acquired stemness after co-culture with M2-TAMs, and resulted in larger tumor size following subcutaneous injection of SMMC-7721 suspended in M2-TAMs supernatants compared with SMMC-7721 alone. Enzyme linked immunosorbent assay showed that TNF-α expression was elevated in supernatants of M2-TAMs and positively correlated with tumor size in the serum of nude mice. Furthermore, we found that the Wnt/β-catenin pathway was a downstream target of TNF-α and that the Wnt/β-catenin inhibitor ICG-001 partially reversed EMT and attenuated cancer stemness. Our results indicate that TNF-α derived from M2-TAMs promote EMT and cancer stemness cells via the Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

Hepatocellular carcinoma (HCC) is one common malignancy. The authors previously demonstrated that miR-570 regulates the development of HCC. This study detected the effect of miR-570 on cell apoptosis, angiogenesis, T cell activation, and proliferation in a tumorigenicity assay in nude mice. miR-570 mimics and negative control (NC) were transfected into SMMC7721 cells, and then, the cells were subcutaneously injected in the right flank in nude mice. Six weeks later, the dissected tumors and peripheral blood were collected. Tumor weight and volume were measured, and expression of miR-570 and apoptosis-related gene Bax/Bcl-2 was detected by quantitative real-time polymerase chain reaction. Hematoxylin and eosin, immunohistochemistry of CD31 and vascular endothelial growth factor (VEGF), TUNEL assay, and flow cytometry detection of CD4 and CD8 in peripheral blood were performed. miR-570 mimics suppressed tumor growth compared with the NC, with decreases in tumor weight and tumor volume. Very few CD31 and VEGF were found in tumor sections in miR-570 mimics group. Bax level was significantly increased, while Bcl-2 level was significantly downregulated. Significant lower ratio of CD3+CD4+ T cells and higher ratio of CD8+IFN-γ+ T cells were found in peripheral blood and tumor tissues in miR-570 mimics than NC. Collectively, miR-570 plays an important role in the proliferation, angiogenesis, and immune escape of HCC, which might be potential diagnostic and predictive biomarkers. [ABSTRACT FROM AUTHOR]

Transcription elongation cycle (TEC) of RNA polymerase II (Pol II) is a process of adding a nucleoside triphosphate to the growing messenger RNA chain. Due to the long timescale events in Pol II TEC, an advanced computational technique, such as Markov State Model (MSM), is needed to provide atomistic mechanism and reaction rates. The combination of MSM and experimental results can be used to build a kinetic network model (KNM) of the whole TEC. This review provides a brief protocol to build MSM and KNM of the whole TEC, along with the latest findings of MSM and other computational studies of Pol II TEC. Lastly, we offer a perspective on potentially using a sequence dependent KNM to predict genome-wide transcription error. [ABSTRACT FROM AUTHOR]

Luminogens with aggregation-induced emission characteristics (AIEgens) are intriguing due to its rapid expansion in various high-tech applications. However, there is still in high demand on the development of novel AIEgens with easy preparation and functionalization, stable structures, tunable emissions, and high quantum efficiency. In this contribution, three AIEgens based on diphenyl isoquinolinium (IQ) derivatives are reported. They can be facilely synthesized and possess high structural stability, favorable visible light excitation, large Stokes shifts, high quantum yields, tunable colors, and sufficient two-photon absorption of near-infrared light. Importantly, they exhibit multifunctionalities. They exhibit mechanochromic property, making them capable to be applied for rewritable papers. They can also be applied in mitochondrial imaging with high specificity, cell permeability, brightness, biocompatibility, and photostability. They are promising for the applications in evaluation of mitochondrial membrane potential and image-guided cancer cell ablation. Last, they are able to stain bacteria in a wash-free manner. All these intriguing results suggest such readily accessible and multifunctional diphenyl IQ-based AIEgens provide a new platform for construction of advanced materials for practical applications. [ABSTRACT FROM AUTHOR]

At the core of RNA interference, the Argonaute proteins (Ago) load and utilize small guide nucleic acids to silence mRNAs or cleave foreign nucleic acids in a sequence specific manner. In recent years, based on extensive structural studies of Ago and its interaction with the nucleic acids, considerable progress has been made to reveal the dynamic aspects of various Ago-mediated processes. Here we review these novel insights into the guide-strand loading, duplex unwinding, and effects of seed mismatch, with a focus on two representative Agos, the human Ago 2 (hAgo2) and the bacterial Thermus thermophilus Ago (TtAgo). In particular, comprehensive molecular simulation studies revealed that although sharing similar overall structures, the two Agos have vastly different conformational landscapes and guide-strand loading mechanisms because of the distinct rigidity of their L1-PAZ hinge. Given the central role of the PAZ motions in regulating the exposure of the nucleic acid binding channel, these findings exemplify the importance of protein motions in distinguishing the overlapping, yet distinct, mechanisms of Ago-mediated processes in different organisms. [ABSTRACT FROM AUTHOR]

A family of six homologous subunits, Mcm2, -3, -4, -5, -6, and -7, each with its own unique features, forms the catalytic core of the eukaryotic replicative helicase. The necessity of six similar but non-identical subunits has been a mystery since its initial discovery. Recent cryo-EM structures of the Mcm2–7 (MCM) double hexamer, its precursors, and the origin recognition complex (ORC)-Cdc6-Cdt1-Mcm2–7 (OCCM) intermediate showed that each of these subunits plays a distinct role in orchestrating the assembly of the pre-replication complex (pre-RC) by ORC-Cdc6 and Cdt1. [ABSTRACT FROM AUTHOR]

We present an efficient density-based adaptive-resolution clustering method APLoD for analyzing large-scale molecular dynamics (MD) trajectories. APLoD performs the k-nearest-neighbors search to estimate the density of MD conformations in a local fashion, which can group MD conformations in the same high-density region into a cluster. APLoD greatly improves the popular density peaks algorithm by reducing the running time and the memory usage by 2-3 orders of magnitude for systems ranging from alanine dipeptide to a 370-residue Maltose-binding protein. In addition, we demonstrate that APLoD can produce clusters with various sizes that are adaptive to the underlying density (i.e., larger clusters at low-density regions, while smaller clusters at high-density regions), which is a clear advantage over other popular clustering algorithms including k-centers and k-medoids. We anticipate that APLoD can be widely applied to split ultra-large MD datasets containing millions of conformations for subsequent construction of Markov State Models. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

High glucose plays a critical role in diabetes. However, the point when high glucose induces diabetes and the organ that triggers the initiation of diabetes remain to be elucidated. The aim of the present study was to clarify the damage induced on different organs of rats, when administered a 2-week infusion of dietary glucose. SD rats (12 weeks old) were randomly divided into normal diet, high glucose infusion (IHG) and oral high glucose (OHG) groups. The levels of fasting blood sugar, tumor necrosis factor (TNF)-α and interleukin (IL)-6 were assessed. Intestine, kidney and liver samples were collected for pathological examination. Feces were collected from the rats for gut microbiota assessment. The results indicated that short-term high glucose induced hyperglycemia that lasted for at least 2 weeks after cessation of high glucose intake. Short-term high glucose also clearly increased the serum levels of IL-6 and TNF-α, led to jejunum mucosa injury and obvious steatosis in hepatocytes, and disturbed the balance of the gut microbiota. OHG led to swelling and necrosis of individual intestinal villi. IHG led to the necrosis and disappearance of cells in the upper layer of the intestinal mucosa. The lesions were confined to the mucosa. A degree of glomerular cell swelling and apoptosis were also observed. Short-term high glucose intake induced lesions in the liver, kidney and intestine, disturbed the balance of the gut microbiota and may consequently induce diabetes complications. [ABSTRACT FROM AUTHOR]

The glutamine binding protein (GlnBP) binds l-glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo- and holo-GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single-molecule FRET techniques to decipher the conformational dynamics of apo-GlnBP. The NMR residual dipolar couplings of apo-GlnBP were in good agreement with a MD-derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four-state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP. [ABSTRACT FROM AUTHOR]

The glutamine binding protein (GlnBP) binds l-glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo- and holo-GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single-molecule FRET techniques to decipher the conformational dynamics of apo-GlnBP. The NMR residual dipolar couplings of apo-GlnBP were in good agreement with a MD-derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four-state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP. [ABSTRACT FROM AUTHOR]

• We propose a three-stage clustering framework based on the pre-computed k nearest neighbors matrix. • A CD metric is developed in this paper to quantify the possibility of each data point as a boundary point. • The BS strategy completes the allocation of labels by shrinking the boundary points inwards. Locating the centers before assigning clustering labels is a traditional routine of clustering methods, which also limits the development of new clustering ideas. In this paper, we achieve the clustering task by firstly identifying the boundary points in the feature space, and then we shrink the boundary points to allocate the un-clustered points. Concretely, we propose a Centroid Drift (CD) metric and a Boundary Shrinkage (BS) strategy to detect boundary points in the feature space and allocate labels for un-clustered points, respectively. Both the CD and BS are closely related to the pre-computed k-nearest neighbor matrix, contributing to the decrease of algorithm parameters. Moreover, the common problems of noise points and non-uniform density distribution of data points in clustering task can also be alleviated with our proposed large value suppression and normalization of k-nearest neighbor distance techniques. The experiments on synthetic datasets, real-world face image datasets and hyperspectral images demonstrate the superiorities of our proposed clustering framework. [ABSTRACT FROM AUTHOR]

Molecules with Möbius topology have drawn increasing attention from scientists in a variety of fields, such as organic chemistry, inorganic chemistry, and material science. However, synthetic difficulties and the lack of functionality impede their fundamental understanding and practical applications. Here, we report the facile synthesis of an aggregation-induced-emission (AIE)-active macrocycle (TPE-ET) and investigate its analogous triply and singly twisted Möbius topologies. Because of the twisted and flexible nature of the tetraphenylethene units, the macrocycle adjusts its conformations so as to accommodate different guest molecules in its crystals. Moreover, theoretical studies including topological and electronic calculations reveal the energetically favorable interconversion process between triply and singly twisted topologies. [ABSTRACT FROM AUTHOR]

Considerable evidence implicates myeloid-derived suppressor cells (MDSCs) promote tumor progression and drug resistance. Sorafenib is the standard first-line therapy for advanced hepatocellular carcinoma (HCC). Clinical evidence indicates that sorafenib resistance is associated with increased MDSCs, by which MDSCs exerts these effects is obscure. This study aimed to investigate the mechanism of sorafenib resistance mediated by MDSCs. A syngeneic mouse-liver cancer cell line BNL was subcutaneously injected to build a tumor-bearing mouse model, and syngeneic MDSCs were adoptive transferred into the tumor-bearing mouse. Tumor tissue was obtained, and transcriptomic analysis of the tumor was carried out on RNAseq data. A coculture system was used to verify the crosstalk between MDSCs and BNL cells. Adoptive MDSCs transfer into tumor-bearing mice induced an increase of tumor-infiltrating MDSCs, which led to tumor growth and impaired antitumor activity of sorafenib in BNL HCC models. MDSCs transfer contributed to tumor fibrosis and tumor-associated fibroblast (CAF) activation, associated with fibroblast growth factor (FGF1) upregulation. In contrast, MDSC depletion by anti-Ly6G+ reduced fibrosis and increased sorafenib antitumor efficacy. Intriguingly, tumor-infiltrating MDSCs barely expressed FGF1. IL-6 derived from MDSCs increased FGF1 expression in BNL liver cancer cells, and anti-IL-6 attenuated this effect in vitro. MAPK pathway, one of the sorafenib targets, is the downstream signaling of FGF1 and is reactivated by MDSCs-mediated FGF1 upregulation. Our finding demonstrated that MDSCs led to tumor growth and sorafenib resistance via FGF1 upregulation and subsequent indirect CAF activation. We offered a novel mechanism of MDSCs-driven HCC progression and sorafenib resistance. [ABSTRACT FROM AUTHOR]

Argonaute (Ago) proteins and microRNAs (miRNAs) are central components in RNA interference, which is a key cellular mechanism for sequence-specific gene silencing. Despite intensive studies, molecular mechanisms of how Ago recognizes miRNA remain largely elusive. In this study, we propose a two-step mechanism for this molecular recognition: selective binding followed by structural re-arrangement. Our model is based on the results of a combination of Markov State Models (MSMs), large-scale protein-RNA docking, and molecular dynamics (MD) simulations. Using MSMs, we identify an open state of apo human Ago-2 in fast equilibrium with partially open and closed states. Conformations in this open state are distinguished by their largely exposed binding grooves that can geometrically accommodate miRNA as indicated in our protein-RNA docking studies. miRNA may then selectively bind to these open conformations. Upon the initial binding, the complex may perform further structural re-arrangement as shown in our MD simulations and eventually reach the stable binary complex structure. Our results provide novel insights in Ago-miRNA recognition mechanisms and our methodology holds great potential to be widely applied in the studies of other important molecular recognition systems. [ABSTRACT FROM AUTHOR]

The RNA polymerase II (Pol II) is a eukaryotic enzyme that catalyzes the synthesis of the messenger RNA using a DNA template. Despite numerous biochemical and biophysical studies, it remains elusive whether the “secondary channel” is the only route for NTP to reach the active site of the enzyme or if the “main channel” could be an alternative. On this regard, crystallographic structures of Pol II have been extremely useful to understand the structural basis of transcription, however, the conformation of the unpaired non-template DNA part of the full transcription bubble (TB) is still unknown. Since diffusion routes of the nucleoside triphosphate (NTP) substrate through the main channel might overlap with the TB region, gaining structural information of the full TB is critical for a complete understanding of Pol II transcription process. In this study, we have built a structural model of Pol II with a complete transcription bubble based on multiple sources of existing structural data and used Molecular Dynamics (MD) simulations together with structural analysis to shed light on NTP entry pathways. Interestingly, we found that although both channels have enough space to allow NTP loading, the percentage of MD conformations containing enough space for NTP loading through the secondary channel is twice higher than that of the main channel. Further energetic study based on MD simulations with NTP loaded in the channels has revealed that the diffusion of the NTP through the main channel is greatly disfavored by electrostatic repulsion between the NTP and the highly negatively charged backbones of nucleotides in the non-template DNA strand. Taken together, our results suggest that the secondary channel is the major route for NTP entry during Pol II transcription. [ABSTRACT FROM AUTHOR]

Organic luminescent materials carryingno phenyl rings have attractedmuch interest from researchers due to their excellent biocompatibilityand good biodegradability, which make them available for potentialapplications in a variety of biomedical areas, such as fluorescentbioprobe, drug delivery and gene carrier, and provide a new insightinto the photophysical process of light emission. In this work, westudied the optical properties of poly[(maleic anhydride)-alt-(vinyl acetate)] (PMV), a pure oxygenic nonconjugatedpolymer and proved that the origin of its emission was associatedwith the clustering of the locked carbonyl groups. PMV exhibits solvatochromism:after interaction with electron-rich solvents, its absorption andemission shift to the longer wavelength region due to the formationof polymer/solvent complexes. This enables fine-tuning of its opticalproperty by varying the solvent without the need of changing the chromophore. [ABSTRACT FROM AUTHOR]

Protein-ligand recognition plays key roles in many biological processes. One of the most fascinating questions about protein-ligand recognition is to understand its underlying mechanism, which often results from a combination of induced fit and conformational selection. In this study, we have developed a three-pronged approach of Markov State Models, Molecular Dynamics simulations, and flux analysis to determine the contribution of each model. Using this approach, we have quantified the recognition mechanism of the choline binding protein (ChoX) to be ∼90% conformational selection dominant under experimental conditions. This is achieved by recovering all the necessary parameters for the flux analysis in combination with available experimental data. Our results also suggest that ChoX has several metastable conformational states, of which an apo-closed state is dominant, consistent with previous experimental findings. Our methodology holds great potential to be widely applied to understand recognition mechanisms underlining many fundamental biological processes. [ABSTRACT FROM AUTHOR]

RNA polymerase is the enzyme that synthesizes RNA during the transcription process. To understand its mechanism, structural studies have provided us pictures of the series of steps necessary to add a new nucleotide to the nascent RNA chain, the steps altogether known as the nucleotide addition cycle (NAC). However, these static snapshots do not provide dynamic information of these processes involved in NAC, such as the conformational changes of the protein and the atomistic details of the catalysis. Computational studies have made efforts to fill these knowledge gaps. In this review, we provide examples of different computational approaches that have improved our understanding of the transcription elongation process for RNA polymerase, such as normal mode analysis, molecular dynamic (MD) simulations, Markov state models (MSMs). We also point out some unsolved questions that could be addressed using computational tools in the future. The dynamics of the different steps in the transcription elongation cycle (EC) of RNA polymerase remain elusive. Here we show how computational tools, such as Normal Mode Analysis, QM/MM and Markov State Models, have improved our understanding of the EC. We also indicate how computational tools can be used to address other unsolved questions of the EC. [ABSTRACT FROM AUTHOR]

The dynamics of the PPi release during the transcription elongation of bacterial RNA polymerase and its effects on the Trigger Loop (TL) opening motion are still elusive. Here, we built a Markov State Model (MSM) from extensive all-atom molecular dynamics (MD) simulations to investigate the mechanism of the PPi release. Our MSM has identified a simple twostate mechanism for the PPi release instead of a more complex four-state mechanism observed in RNA polymerase II (Pol II). We observed that the PPi release in bacterial RNA polymerase occurs at sub-microsecond timescale, which is, 3-fold faster than that in Pol II. After escaping from the active site, the (Mg-PPi)2- group passes through a single elongated metastable region where several positively charged residues on the secondary channel provide favorable interactions. Surprisingly, we found that the PPi release is not coupled with the TL unfolding but correlates tightly with the side-chain rotation of the TL residue R1239. Our work sheds light on the dynamics underlying the transcription elongation of the bacterial RNA polymerase. [ABSTRACT FROM AUTHOR]

We implemented a GPU-powered parallel k-centers algorithm to perform clustering on the conformations of molecular dynamics (MD) simulations. The algorithm is up to two orders of magnitude faster than the CPU implementation. We tested our algorithm on four protein MD simulation datasets ranging from the small Alanine Dipeptide to a 370-residue Maltose Binding Protein (MBP). It is capable of grouping 250,000 conformations of the MBP into 4000 clusters within 40 seconds. To achieve this, we effectively parallelized the code on the GPU and utilize the triangle inequality of metric spaces. Furthermore, the algorithm's running time is linear with respect to the number of cluster centers. In addition, we found the triangle inequality to be less effective in higher dimensions and provide a mathematical rationale. Finally, using Alanine Dipeptide as an example, we show a strong correlation between cluster populations resulting from the k-centers algorithm and the underlying density. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Vibrationally resolved fluorescence spectra of the β-hairpintrpzip2 peptide at two temperatures as well as during a T-jump unfolding process are simulated on the basis of a combinationof Markov state models and quantum chemistry schemes. The broad asymmetricspectral line shape feature is reproduced by considering the exciton–phononcouplings. The temperature dependent red shift observed in the experimenthas been attributed to the state population changes of specific chromophores.Through further theoretical study, it is found that both the environment'selectric field and the chromophores’ geometry distortions areresponsible for tryptophan fluorescence shift. [ABSTRACT FROM AUTHOR]

We present a set of force field (FF) parameters compatible with the AMBER03 FF to describe five cofactors in photosystem II (PSII) of oxygenic photosynthetic organisms: plastoquinone-9 (three redox forms), chlorophyll-a, pheophytin-a, heme-b, and β-carotene. The development of a reliable FF for these cofactors is an essential step for performing molecular dynamics simulations of PSII. Such simulations are important for the calculation of absorption spectrum and the further investigation of the electron and energy transfer processes. We have derived parameters for partial charges, bonds, angles, and dihedral-angles from solid theoretical models using systematic quantum mechanics (QM) calculations. We have shown that the developed FF parameters are in good agreement with both ab initio QM and experimental structural data in small molecule crystals as well as protein complexes. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In this article, we review the recent progress in multiresolution modeling of structure and dynamics of protein, RNA and their complexes. Many approaches using both physics-based and knowledge-based potentials have been developed at multiple granularities to model both protein and RNA. Coarse graining can be achieved not only in the length, but also in the time domain using discrete time and discrete state kinetic network models. Models with different resolutions can be combined either in a sequential or parallel fashion. Similarly, the modeling of assemblies is also often achieved using multiple granularities. The progress shows that a multiresolution approach has considerable potential to continue extending the length and time scales of macromolecular modeling. [ABSTRACT FROM PUBLISHER]

We proposed a computational protocol of simulating the T-jump peptide unfolding experiments and the related transient IR and two-dimensional IR (2DIR) spectra based on the Markov state model (MSM) and nonlinear exciton propagation (NEP) methods. MSMs partition the conformation space into a set of nonoverlapping metastable states, and we can calculate spectra signal for each of these states using the NEP method. Thus the overall spectroscopic observable for a given system is simply the sum of spectra of different metastable states weighted by their populations. We show that results from MSMs constructed from a large number of simulations have a much better agreement with the equilibrium experimental 2DIR spectra compared to that generated from straightforward MD simulations starting from the folded state. This indicates that a sufficient sampling of important relevant conformational states is critical for calculating the accurate spectroscopic observables. MSMs are also capable of simulating the unfolding relaxation dynamics upon the temperature jump. The agreement of the simulation using MSMs and NEP with the experiment not only provides a justification for our protocol, but also provides the physical insight of the underlying spectroscopic observables. The protocol we developed has the potential to be extended to simulate a wide range of fast triggering plus optical detection experiments for biomolecules. [ABSTRACT FROM AUTHOR]

Favipiravir (T-705) has been developed as a potent anti-influenza drug and exhibited a strong inhibition effect against a broad spectrum of RNA viruses. Its active form, ribofuranosyl-triphosphate (T-705-RTP), functions as a competitive substrate for the RNA-dependent RNA polymerase (RdRp) of the influenza A virus (IAV). However, the exact inhibitory mechanisms of T-705 remain elusive and subject to a long-standing debate. Although T-705 has been proposed to inhibit transcription by acting as a chain terminator, it is also paradoxically suggested to be a mutagen towards IAV RdRp by inducing mutations due to its ambiguous base pairing of C and U. Here, we combined biochemical assay with molecular dynamics (MD) simulations to elucidate the molecular mechanism underlying the inhibitory functions exerted by T-705 in IAV RdRp. Our in vitro transcription assay illustrated that IAV RdRp could recognize T-705 as a purine analogue and incorporate it into the nascent RNA strand. Incorporating a single T-705 is incapable of inhibiting transcription as extra natural nucleotides can be progressively added. However, when two consecutive T-705 are incorporated, viral transcription is completely terminated. MD simulations reveal that the sequential appearance of two T-705 in the nascent strand destabilizes the active site and disrupts the base stacking of the nascent RNA. Altogether, our results provide a plausible explanation for the inhibitory roles of T-705 targeting IAV RdRp by integrating the computational and experimental methods. Our study also offers a comprehensive platform to investigate the inhibition effect of antivirals and a novel explanation for the designing of anti-flu drugs. [Display omitted] • Single T-705 could be incorporated by flu RdRp. • Double T-705 choke the transcription of flu RdRp. • T-705 inhibits polyadenylation. [ABSTRACT FROM AUTHOR]

The H19 long non-coding RNA is involved in the development of tamoxifen resistance in breast cancer. However, the relationship between H19 and the metastatic potential and treatment options for tamoxifen-resistant (TAMR) breast cancer is not completely understood. Curcumin inhibits cellular proliferation, migration and invasiveness in several cancer types, including pancreatic cancer, breast cancer and chronic myeloid leukemia. The present study aimed to investigate the role of H19 in MCF-7/TAMR cell epithelial-mesenchymal transition (EMT), migration and invasiveness, and to assess the ability of curcumin to inhibit H19-mediated effects. Reverse transcription-quantitative PCR and western blot analysis were conducted to detect the gene or protein expression. Cell Counting Kit-8, wound healing and Transwell invasion assays were performed to estimate the capabilities of cell viability, invasion and migration. H19 overexpression enhanced MCF-7/TAMR cell EMT, invasion and migration by upregulating Snail. Furthermore, curcumin notably decreased the expression levels of epithelial marker E-cadherin and markedly increased the expression levels of mesenchymal marker N-cadherin in MCF-7/TAMR cells compared with the control group. In addition, following treatment with curcumin for 48 h, H19 expression was decreased in a dose-dependent manner. Moreover, curcumin treatment for 48 h significantly attenuated H19-induced alterations in N-cadherin and E-cadherin expression levels. Curcumin also prevented H19-induced invasion and migration. The present study indicated that H19 may serve as a promoting factor of EMT, invasion and migration in MCF-7/TAMR cells, suggesting that curcumin may prevent H19-associated metastasis. Therefore, curcumin may serve as a promising therapeutic drug for patients with TAMR breast cancer. [ABSTRACT FROM AUTHOR]

Markov state models (MSMs) have become a popular approach for investigating the conformational dynamics of proteins and other biomolecules. MSMs are typically built from numerous molecular dynamics simulations by dividing the sampled configurations into a large number of microstates based on geometric criteria. The resulting microstate model can then be coarse-grained into a more understandable macrostate model by lumping together rapidly mixing microstates into larger, metastable aggregates. However, finite sampling often results in the creation of many poorly sampled microstates. During coarse-graining, these states are mistakenly identified as being kinetically important because transitions to/from them appear to be slow. In this paper, we propose a formalism based on an algebraic principle for matrix approximation, i.e., the Nyström method, to deal with such poorly sampled microstates. Our scheme builds a hierarchy of microstates from high to low populations and progressively applies spectral clustering on sets of microstates within each level of the hierarchy. It helps spectral clustering identify metastable aggregates with highly populated microstates rather than being distracted by lowly populated states. We demonstrate the ability of this algorithm to discover the major metastable states on two model systems, the alanine dipeptide and trpzip2 peptide. [ABSTRACT FROM AUTHOR]

An escalating pandemic by the novel SARS-CoV-2 virus is impacting global health and effective therapeutic options are urgently needed. We evaluated the in vitro antiviral effect of compounds that were previously reported to inhibit coronavirus replication and compounds that are currently under evaluation in clinical trials for SARS-CoV-2 patients. We report the antiviral effect of remdesivir, lopinavir, homorringtonine, and emetine against SARS-CoV-2 virus in Vero E6 cells with the estimated 50% effective concentration at 23.15 μM, 26.63 μM, 2.55 μM and 0.46 μM, respectively. Ribavirin or favipiravir that are currently evaluated under clinical trials showed no inhibition at 100 μM. Synergy between remdesivir and emetine was observed, and remdesivir at 6.25 μM in combination with emetine at 0.195 μM may achieve 64.9% inhibition in viral yield. Combinational therapy may help to reduce the effective concentration of compounds below the therapeutic plasma concentrations and provide better clinical benefits. • Remdesivir inhibits SARS-CoV-2 replication in Vero-E6 cells with EC 50 at 23.15 μM. • Lopinavir but not ritonavir inhibits SARS-CoV-2 replication with EC 50 at 26.63 μM. • Homoharringtonine and emetine inhibits SARS-CoV-2 replication with EC 50 at 2.55 and 0.46 μM, respectively. • Combination of remdesivir and emetine showed synergistic effect in vitro. [ABSTRACT FROM AUTHOR]

An improved anti-noise morphology vision navigation algorithm is proposed for intelligent tractor tillage in a complex agricultural field environment. At first, the two key steps of guided filtering and improved anti-noise morphology navigation line extraction were addressed in detail. Then, the experiments were carried out in order to verify the effectiveness and advancement of the presented algorithm. Finally, the optimal template and its application condition were studied for improving the image-processing speed. The comparison experiment results show that the YCbCr color space has minimum time consumption of 0.094   s in comparison with HSV, HIS, and 2R-G-B color spaces. The guided filtering method can effectively distinguish the boundary between the tillage soil compared to other competing vanilla methods such as Tarel, multi-scale retinex, wavelet-based retinex, and homomorphic filtering in spite of having the fastest processing speed of 0.113   s . The extracted soil boundary line of the improved anti-noise morphology algorithm has the best precision and speed compared to other operators such as Sobel, Roberts, Prewitt, and Log. After comparing different sizes of image templates, the optimal template with the size of 140   ×   260 pixels could achieve high-precision vision navigation while the course deviation angle was not more than 7.5 ° . The maximum tractor speed of the optimal template and global template were 51.41   km / h and 27.47   km / h , respectively, which can meet the real-time vision navigation requirement of the smart tractor tillage operation in the field. The experimental vision navigation results demonstrated the feasibility of the autonomous vision navigation for tractor tillage operation in the field using the tillage soil boundary line extracted by the proposed improved anti-noise morphology algorithm, which has broad application prospect. [ABSTRACT FROM AUTHOR]

Hospital‐acquired infection causes many deaths worldwide and calls for the urgent need for antibacterial biomaterials used in clinic that can selectively kill harmful bacteria. The present study rationally designs fusion peptides capable of undergoing 2D self‐assembly on the poly(methyl methacrylate) surface to form a smart surface, which can maintain a desirable orientation via electrostatic interactions. The in vitro assay shows that the smart surface can recognize bacteria to exert antibacterial activity and is nontoxic toward mouse bone mesenchymal stem cells. Excitingly, the smart surface can distinguish different bacterial strains. This selective feature, from being broad‐spectrum to being highly selective against S. aureus, can be altered by varying the number of amino acids in the recognition sequences. By all‐atom molecular dynamics simulations, it is also found that the recognition sequence in the peptide is critical for the selectivity toward specific bacterial strains, in which a less accessible surface area for the bacteria in the antimicrobial peptide sequence is responsible for such selectivity. Finally, the smart surface can inhibit S. aureus infection in vivo with much more rapid tissue‐healing compared to the control. [ABSTRACT FROM AUTHOR]

Summary DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP–DksA complex. The structural data support a model for transcriptional inhibition in which ppGpp potentiates the destabilization of open complexes by DksA. This work establishes a structural basis for understanding the pleiotropic effects of DksA and ppGpp on transcriptional regulation in proteobacteria. [ABSTRACT FROM AUTHOR]

Summary Several unconventional myosins contain a highly charged single α helix (SAH) immediately following the calmodulin (CaM) binding IQ motifs, functioning to extend lever arms of these myosins. How such SAH is connected to the IQ motifs and whether the conformation of the IQ motifs-SAH segments are regulated by Ca 2+ fluctuations are not known. Here, we demonstrate by solving its crystal structure that the predicted SAH of myosin VIIa (Myo7a) forms a stable SAH. The structure of Myo7a IQ5-SAH segment in complex with apo-CaM reveals that the SAH sequence can extend the length of the Myo7a lever arm. Although Ca 2+ -CaM remains bound to IQ5-SAH, the Ca 2+ -induced CaM binding mode change softens the conformation of the IQ5-SAH junction, revealing a Ca 2+ -induced lever arm flexibility change for Myo7a. We further demonstrate that the last IQ motif of several other myosins also binds to both apo- and Ca 2+ -CaM, suggesting a common Ca 2+ -induced conformational regulation mechanism. [ABSTRACT FROM AUTHOR]

Thalassospiramides comprise a large family of lipopeptide natural products produced by Thalassospira and Tistrella marine bacteria. Here we provide further evidence of their nanomolar inhibitory activity against the human calpain 1 protease. Analysis of structure-activity relationship data supported our hypothesis that the rigid 12-membered ring containing an α,β-unsaturated carbonyl moiety is the pharmacologically active functional group, in contrast to classic electrophilic 'warheads' in known calpain inhibitors. Using a combination of chemical modifications, mass spectrometric techniques, site-directed mutagenesis, and molecular modeling, we show the covalent binding of thalassospiramide's α,β-unsaturated carbonyl moiety to the thiol group of calpain's catalytic Cys115 residue by a Michael 1,4-addition reaction. As nanomolar calpain inhibitors with promising selectivity and low toxicity from natural sources are rare, we consider thalassospiramides as promising drug leads. [ABSTRACT FROM AUTHOR]