Infrared Spectra of Hydrogen-Bonded Molecular Complexes Under Spatial Confinement

Infrared (IR) spectroscopy is commonly used in chemical laboratories to study the geometrical structure of molecules and molecular complexes. The analysis of experimental IR spectra can nowadays be reliably supported by the results of quantum-chemical computations as vibrational frequencies and corresponding vibrational transition intensities are routinely calculated using harmonic approximation by virtually all quantum chemistry packages. In the present study we combine the methodology of computing vibrational spectra using high-level electron correlation treatments with an analytical potential-based approach to take into account spatial confinement effects. Using this approach, we perform a pioneering analysis of the impact of the spatial confinement caused by a cylindrical harmonic oscillator potential on the harmonic vibrational transition intensities and frequencies of two hydrogen-bonded complexes: HCN…HCN and HCN…HNC. The emphasis is put on the largest-intensity bands, which correspond to the stretching vibrations. The obtained results demonstrate that embedding the molecular complexes in an external confining potential causes significant changes of transition intensities and vibrational frequencies.

fingeRNAt - a novel tool for high-throughput analysis of nucleic acid-ligand interactions

Computational methods play a pivotal role in drug discovery and are widely applied in virtual screening, structure optimization, and compound activity profiling. Over the last decades, almost all the attention in medicinal chemistry has been directed to protein-ligand binding, and computational tools have been created with this target in mind. With novel discoveries of functional RNAs and their possible applications, RNAs have gained considerable attention as potential drug targets. However, the availability of bioinformatics tools for nucleic acids is limited. Here, we introduce fingeRNAt - a software tool for detecting non-covalent interactions formed in complexes of nucleic acids with ligands. The program detects nine types of interactions: (i) hydrogen and (ii) halogen bonds, (iii) cation-anion, (iv) pi-cation, (v) pi-anion, (vi) pi-stacking, (vii) inorganic ion-mediated, (viii) water-mediated, and (ix) lipophilic interactions. However, the scope of detected interactions can be easily expanded using a simple plugin system. In addition, detected interactions can be visualized using the associated PyMOL plugin, which facilitates the analysis of medium-throughput molecular complexes. Interactions are also encoded and stored as a bioinformatics-friendly Structural Interaction Fingerprint (SIFt) - a binary string where the respective bit in the fingerprint is set to 1 if a particular interaction is present and to 0 otherwise. This output format, in turn, enables high-throughput analysis of interaction data using data analysis techniques. We present applications of fingeRNAt-generated interaction fingerprints for visual and computational analysis of RNA-ligand complexes, including analysis of interactions formed in experimentally determined RNA-small molecule ligand complexes deposited in the Protein Data Bank. We propose interaction-based similarity based on fingerprints as an alternative measure to RMSD to recapitulate complexes with similar interactions but different folding. We present an application of molecular fingerprints for the clustering of molecular complexes. This approach can be used to group ligands that form similar binding networks and thus have similar biological properties.

Proteomic analysis identifies ZMYM2 as endogenous binding partner of TBX18 protein in 293 and A549 cells

The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development.

Generation of Dystrophin Short Product-Specific Tag-Insertion Mouse: Distinct Dp71 Glycoprotein Complexes at Inhibitory Postsynapse and Glia Limitans

Duchenne muscular dystrophy (DMD), the most severe form of dystrophinopathies, is a fatal X-linked recessive neuromuscular disorder characterized by progressive muscle degeneration and various extents of intellectual disabilities. Physiological and pathological roles of the responsible gene, dystrophin, in the brain remain elusive due to the presence of multiple dystrophin products, mainly full-length dystrophin, Dp427, and the short product, Dp71. In this study, we generated a Dp71-specific hemagglutinin (HA) peptide tag-insertion mice to enable specific detection of intrinsic Dp71 expression by anti-HA tag antibodies. Immunohistochemical detections in the transgenic mice demonstrated Dp71 expression not only at the blood-brain barrier, where astrocytic endfeet surround the microvessels, but also at the inhibitory postsynapse of hippocampal dentate granule neurons. Interestingly, hippocampal cornu ammonis (CA)1 pyramidal neurons were negative for Dp71 although Dp427 detected by anti-dystrophin antibody was clearly present at the inhibitory postsynapse, suggesting cell-type dependent dystrophin expressions. Precise examination using the primary hippocampal culture validated exclusive localization of Dp71 at the inhibitory postsynaptic compartment but not at the excitatory synapse in neurons. We further performed interactome analysis and found that Dp71 formed distinct molecular complexes, i.e. synapse-associated Dp71 interacted with dystroglycan (Dg) and dystrobrevinb (Dtnb) whereas glia-associated Dp71 did with Dg and dystrobrevina (Dtna). Thus, our data indicates that Dp71 and its binding partners are relevant to the inhibitory postsynaptic function of hippocampal granule neurons and the novel Dp71-transgenic mouse provides a valuable tool to understand precise physiological expressions and functions of Dp71 and its interaction proteins in vivo and in vitro.

Immune Infiltration, Cancer Stemness, and Targeted Therapy in Gastrointestinal Stromal Tumor

ObjectiveTo investigate the characteristics of the tumor immune microenvironment in patients with gastrointestinal stromal tumor (GIST) and identify cancer stem-like properties of GIST to screen potential druggable molecular targets.MethodsThe gene expression data of 60 patients with GIST was retrieved from the Array Express database. CIBERSORT was applied to calculate the level of immune infiltration. ssGSEA and ESTIMATE were used to calculate the cancer stemness index and tissue purity. The Connectivity Map (CMAP) database was implemented to screen targeted drugs based on cancer stem-like properties of GIST.ResultThere was a difference in the level of immune infiltration between the metastasis and non-metastasis GIST groups. The low level of T-cell infiltration was correlated with high tumor purity and tumor stemness index, and the correlation coefficients were -0.87 and -0.61 (p < 0.001), respectively. Furthermore, there was a positive correlation between cancer stemness index and cell purity (p < 0.001). The cancer stemness index in the metastasis group was higher than that in the non-metastasis group (p = 0.0017). After adjusting for tumor purity, there was no significant correlation between T-cell infiltration and cancer stemness index (p = 0.086). Through the pharmacological mechanism of topoisomerase inhibitors, six molecular complexes may be the targets of GIST treatment.ConclusionImmune infiltration in GIST patients is related to cancer stem-like properties, and the correlation relies on tumor purity. Cancer stemness index can be used as a new predictive biomarker of tumor metastasis and targets of drug therapy for GIST patients.

Forensic determination of determination of damage to human internal organs by the method of reconstruction of the optical activity of histological sections

The article presents the results of experimental testing of the technique of tomographic reconstruction of circular birefringence maps of molecular complexes in a digital histological study of the age of damage to tissues of internal organs (brain, liver and kidney) of temporary monitoring of the mean value, dispersion, asymmetry and kurtosis, characterizing the distribution of the magnitude of the optical activity of histological sections of the brain, liver and kidney identification of the temporal extent of linear changes in statistical parameters and the accuracy of determining the duration of damage to human internal organs by digital histological methods of polarization reconstruction (tomography) of circular birefringence of molecular complexes. Aim of the work. Development of a technique for determining the duration of damage to human internal organs by digital histological methods of polarization reconstruction of circular birefringence of molecular complexes. Materials and methods. The object of the study was the histology of samples of human internal organs (brain, kidney and liver) with different duration of damage from 1 hour to 120 hours. For control, we used BT samples of those who died from coronary artery disease with different duration of damage from 1 hour to 120 hours. The studies were carried out using the technique of polarization reconstruction of circular birefringence of molecular complexes. Results. A set of treatment-relevant relationships between temporal changes in the statistical structure of topographic maps of circular birefringence of optically active molecular complexes of histological sections of human internal organs with different duration of damage and variations in the mean value, dispersion, asymmetry and kurtosis, characterizing the distribution of the value of this parameter of anisotropy, has been determined. Conclusions. A new original method has been developed for tomography of the optical activity of molecular complexes of tissues of human internal organs in a digital histological study of the age of damage to the tissues of the brain, liver and kidney, as well as the myocardium and lung tissue at a time interval of 1 hour. up to 120 hours

Insights into neurodegeneration from electron microscopy studies

Neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, pose an increasingly severe burden for individuals and society in an ageing population. The causes and mechanisms of the diseases are poorly understood and as yet there are no effective treatments. Some of the molecular complexes involved in degeneration have been identified and electron microscopy has provided an essential tool in the investigations. The focus of this review is to show how electron microscopy has contributed historically to the understanding of disease and to summarize the most striking current advances. It does not seek to cover in detail the recent technical developments in microscopy, involving better microscopes, better electron detectors and more powerful image processing techniques, which have made possible the new insights. In many instances pathological filament assemblies are associated with brain cells that die in the disease, causing the observed symptoms such as dementia or movement disorders. Using electron microscopy it is now possible to go beyond morphological descriptions to produce atomic structures of many of the filaments. This information may help to understand the seeding and assembly of the filaments, with the aim of finding small molecule inhibitors that could potentially provide a form of treatment for the diseases.