Precise druggability of the PTH type 1 receptor.

Class B G protein-coupled receptors (GPCRs) are notoriously difficult to target by small molecules because their large orthosteric peptide-binding pocket embedded deep within the transmembrane domain limits the identification and development of nonpeptide small molecule ligands. Using the parathyroid hormone type 1 receptor (PTHR) as a prototypic class B GPCR target, and a combination of molecular dynamics simulations and elastic network model-based methods, we demonstrate that PTHR druggability can be effectively addressed. Here we found a key mechanical site that modulates the collective dynamics of the receptor and used this ensemble of PTHR conformers to identify selective small molecules with strong negative allosteric and biased properties for PTHR signaling in cell and PTH actions in vivo. This study provides a computational pipeline to detect precise druggable sites and identify allosteric modulators of PTHR signaling that could be extended to GPCRs to expedite discoveries of small molecules as novel therapeutic candidates.

Neonatal exposure to sevoflurane induces adolescent neurobehavioral dysfunction by interfering with hippocampal glycerophoslipid metabolism in rats.

Sevoflurane exposure in the neonatal period causes long-term developmental neuropsychological dysfunction, including memory impairment and anxiety-like behaviors. However, the molecular mechanisms underlying such effects have not been fully elucidated. In this study, we investigated the effect of neonatal exposure to sevoflurane on neurobehavioral profiles in adolescent rats, and applied an integrated approach of lipidomics and proteomics to investigate the molecular network implicated in neurobehavioral dysfunction. We found that neonatal exposure to sevoflurane caused cognitive impairment and social behavior deficits in adolescent rats. Lipidomics analyses revealed that sevoflurane significantly remodeled hippocampal lipid metabolism, including lysophatidylcholine (LPC) metabolism, phospholipid carbon chain length and carbon chain saturation. Through a combined proteomics analysis, we found that neonatal exposure to sevoflurane significantly downregulated the expression of lysophosphatidylcholine acyltransferase 1 (LPCAT1), a key enzyme in the regulation of phospholipid metabolism, in the hippocampus of adolescent rats. Importantly, hippocampal LPCAT1 overexpression restored the dysregulated glycerophospholipid (GP) metabolism and alleviated the learning and memory deficits caused by sevoflurane. Collectively, our evidence that neonatal exposure to sevoflurane downregulates LPCAT1 expression and dysregulates GP metabolism in the hippocampus, which may contribute to the neurobehavioral dysfunction in the adolescent rats.

Ancient DNA and multimethod dating confirm the late arrival of anatomically modern humans in southern China.

The expansion of anatomically modern humans (AMHs) from Africa around 65,000 to 45,000 y ago (ca. 65 to 45 ka) led to the establishment of present-day non-African populations. Some paleoanthropologists have argued that fossil discoveries from Huanglong, Zhiren, Luna, and Fuyan caves in southern China indicate one or more prior dispersals, perhaps as early as ca. 120 ka. We investigated the age of the human remains from three of these localities and two additional early AMH sites (Yangjiapo and Sanyou caves, Hubei) by combining ancient DNA (aDNA) analysis with a multimethod geological dating strategy. Although U-Th dating of capping flowstones suggested they lie within the range ca. 168 to 70 ka, analyses of aDNA and direct AMS 14C dating on human teeth from Fuyan and Yangjiapo caves showed they derive from the Holocene. OSL dating of sediments and AMS 14C analysis of mammal teeth and charcoal also demonstrated major discrepancies from the flowstone ages; the difference between them being an order of magnitude or more at most of these localities. Our work highlights the surprisingly complex depositional history recorded at these subtropical caves which involved one or more episodes of erosion and redeposition or intrusion as recently as the late Holocene. In light of our findings, the first appearance datum for AMHs in southern China should probably lie within the timeframe set by molecular data of ca. 50 to 45 ka.

Integrated lipidomic and transcriptomic analysis reveals clarithromycin-induced alteration of glycerophospholipid metabolism in the cerebral cortex of mice.

Clarithromycin (CLA) has been widely used in the treatment of bacterial infection. Research reveals the adverse effects on the central nervous system among patients receiving CLA treatment; whereas, a relevant underlying mechanism remains considerably unclear. According to our research, an integrated lipidomic and transcriptomic analysis was applied to explore the effect of CLA on neurobehavior. CLA treatment caused anxiety-like behaviors dose-dependently during open field as well as elevated plus maze trials on mice. Transcriptomes and LC/MS-MS-based metabolomes were adopted for investigating how CLA affected lipidomic profiling as well as metabolic pathway of the cerebral cortex. CLA exposure greatly disturbed glycerophospholipid metabolism and the carbon chain length of fatty acids. By using whole transcriptome sequencing, we found that CLA significantly downregulated the mRNA expression of CEPT1 and CHPT1, two key enzymes involved in the synthesis of glycerophospholipids, supporting the findings from the lipidomic profiling. Also, CLA causes changes in neuronal morphology and function in vitro, which support the existing findings concerning neurobehavior in vivo. We speculate that altered glycerophospholipid metabolism may be involved in the neurobehavioral effect of CLA. Our findings contribute to understanding the mechanisms of CLA-induced adverse effects on the central nervous system. 1. Clarithromycin treatment caused anxiety-like behavior with dose-dependent response both in the open field and elevated plus maze test in mice; 2. Clarithromycin exposing predominately disturbed the metabolism of glycerophospholipids in the cerebral cortex of mice; 3. Clarithromycin application remarkably attenuated CEPT1 and CHPT1 gene expression, which participate in the last step in the synthesis of glycerophospholipids; 4. The altered glycerophospholipid metabolomics may be involved in the abnormal neurobehavior caused by clarithromycin.

4R Tau Modulates Cocaine-Associated Memory through Adult Dorsal Hippocampal Neurogenesis.

The development, persistence and relapse of drug addiction require drug memory that generally develops with drug administration-paired contextual stimuli. Adult hippocampal neurogenesis (AHN) contributes to cocaine memory formation; however, the underlying mechanism remains unclear. Male mice hippocampal expression of Tau was significantly decreased during the cocaine-associated memory formation. Genetic overexpression of four microtubule-binding repeats Tau (4R Tau) in the mice hippocampus disrupted cocaine memory by suppressing AHN. Furthermore, 4R Tau directly interacted with phosphoinositide 3-kinase (PI3K)-p85 and impaired its nuclear translocation and PI3K-AKT signaling, processes required for hippocampal neuron proliferation. Collectively, 4R Tau modulates cocaine memory formation by disrupting AHN, suggesting a novel mechanism underlying cocaine memory formation and provide a new strategy for the treatment of cocaine addiction.SIGNIFICANCE STATEMENT Drug memory that generally develops with drug-paired contextual stimuli and drug administration is critical for the development, persistence and relapse of drug addiction. Previous studies have suggested that adult hippocampal neurogenesis (AHN) plays a role in cocaine memory formation. Here, we showed that Tau was significantly downregulated in the hippocampus in the cocaine memory formation. Tau knock-out (KO) promoted AHN in the hippocampal dentate gyrus (DG), resulting in the enhanced memory formation evoked by cocaine-cue stimuli. In contrast, genetically overexpressed 4R Tau in the hippocampus disrupted cocaine-cue memory by suppressing AHN. In addition, 4R Tau interacted directly with phosphoinositide 3-kinase (PI3K)-p85 and hindered its nuclear translocation, eventually repressing PI3K-AKT signaling, which is essential for hippocampal neuronal proliferation.

ProDy 2.0: increased scale and scope after 10 years of protein dynamics modelling with Python.

SUMMARY: ProDy, an integrated application programming interface developed for modelling and analysing protein dynamics, has significantly evolved in recent years in response to the growing data and needs of the computational biology community. We present major developments that led to ProDy 2.0: (i) improved interfacing with databases and parsing new file formats, (ii) SignDy for signature dynamics of protein families, (iii) CryoDy for collective dynamics of supramolecular systems using cryo-EM density maps and (iv) essential site scanning analysis for identifying sites essential to modulating global dynamics. AVAILABILITY AND IMPLEMENTATION: ProDy is open-source and freely available under MIT License from https://github.com/prody/ProDy. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

mTOR regulates cocaine-induced behavioural sensitization through the SynDIG1-GluA2 interaction in the nucleus accumbens.

Behavioral sensitization is a progressive increase in locomotor or stereotypic behaviours in response to drugs. It is believed to contribute to the reinforcing properties of drugs and to play an important role in relapse after cessation of drug abuse. However, the mechanism underlying this behaviour remains poorly understood. In this study, we showed that mTOR signaling was activated during the expression of behavioral sensitization to cocaine and that intraperitoneal or intra-nucleus accumbens (NAc) treatment with rapamycin, a specific mTOR inhibitor, attenuated cocaine-induced behavioural sensitization. Cocaine significantly modified brain lipid profiles in the NAc of cocaine-sensitized mice and markedly elevated the levels of phosphatidylinositol-4-monophosphates (PIPs), including PIP, PIP2, and PIP3. The behavioural effect of cocaine was attenuated by intra-NAc administration of LY294002, an AKT-specific inhibitor, suggesting that PIPs may contribute to mTOR activation in response to cocaine. An RNA-sequencing analysis of the downstream effectors of mTOR signalling revealed that cocaine significantly decreased the expression of SynDIG1, a known substrate of mTOR signalling, and decreased the surface expression of GluA2. In contrast, AAV-mediated SynDIG1 overexpression in NAc attenuated intracellular GluA2 internalization by promoting the SynDIG1-GluA2 interaction, thus maintaining GluA2 surface expression and repressing cocaine-induced behaviours. In conclusion, NAc SynDIG1 may play a negative regulatory role in cocaine-induced behavioural sensitization by regulating synaptic surface expression of GluA2.

QuartataWeb: Integrated Chemical-Protein-Pathway Mapping for Polypharmacology and Chemogenomics.

SUMMARY: QuartataWeb is a user-friendly server developed for polypharmacological and chemogenomics analyses. Users can easily obtain information on experimentally verified (known) and computationally predicted (new) interactions between 5494 drugs and 2807 human proteins in DrugBank, and between 315 514 chemicals and 9457 human proteins in the STITCH database. In addition, QuartataWeb links targets to KEGG pathways and GO annotations, completing the bridge from drugs/chemicals to function via protein targets and cellular pathways. It allows users to query a series of chemicals, drug combinations or multiple targets, to enable multi-drug, multi-target, multi-pathway analyses, toward facilitating the design of polypharmacological treatments for complex diseases. AVAILABILITY AND IMPLEMENTATION: QuartataWeb is freely accessible at http://quartata.csb.pitt.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The utility of competency-oriented clinical laboratory teaching combined with case-based learning (CBL).

OBJECTIVES: This study aimed to evaluate the effectiveness and efficiency of competency-oriented clinical laboratory teaching combined with case-based learning (CBL) and improve the examination of students' competence of laboratory medicine. METHODS: A total of 107 medical laboratory medicine interns at the Affiliated Hospital of Xuzhou Medical University from June 2017 to July 2019 volunteered to participate in the study and were randomly assigned into a control group with training of the traditional teacher-centered method, and an experimental group under a CBL teaching program. Student basic theory tests and skill assessment were designed to evaluate what the students gained from their internship when they completed their studies at the Affiliated Hospital of Xuzhou Medical University. RESULTS: Compared to students in the control group taught with the teacher-centered method, those in the CBL teaching program had significantly higher theory test scores and skill assessment scores on average. Competencies with particularly significant improvement included identification and processing of instrument alarm information, analysis of test results, identification and solution of the problem, as well as identification and reporting of the critical value and clinical communication. CONCLUSIONS: The competency-oriented teaching method combined with CBL is an effective method for improving students' professional knowledge, increasing language expression, and enhancing interpersonal relationship and teamwork, which is worthy of being promoted in laboratory medicine teaching.

MolADI: A Web Server for Automatic Analysis of Protein-Small Molecule Dynamic Interactions.

Protein-ligand interaction analysis is important for drug discovery and rational protein design. The existing online tools adopt only a single conformation of the complex structure for calculating and displaying the interactions, whereas both protein residues and ligand molecules are flexible to some extent. The interactions evolved with time in the trajectories are of greater interest. MolADI is a user-friendly online tool which analyzes the protein-ligand interactions in detail for either a single structure or a trajectory. Interactions can be viewed easily with both 2D graphs and 3D representations. MolADI is available as a web application.

Modulation of Toroidal Proteins Dynamics in Favor of Functional Mechanisms upon Ligand Binding.

Toroidal proteins serve as molecular machines and play crucial roles in biological processes such as DNA replication and RNA transcription. Despite progress in the structural characterization of several toroidal proteins, we still lack a mechanistic understanding of the significance of their architecture, oligomerization states, and intermolecular interactions in defining their biological function. In this work, we analyze the collective dynamics of toroidal proteins with different oligomerization states, namely, dimeric and trimeric DNA sliding clamps, nucleocapsid proteins (4-, 5-, and 6-mers) and Trp RNA-binding attenuation proteins (11- and 12-mers). We observe common global modes, among which cooperative rolling stands out as a mechanism enabling DNA processivity, and clamshell motions as those underlying the opening/closure of the sliding clamps. Alterations in global dynamics due to complexation with DNA or the clamp loader are shown to assist in enhancing motions to enable robust function. The analysis provides new insights into the differentiation and enhancement of functional motions upon intersubunit and intermolecular interactions.

Shared Signature Dynamics Tempered by Local Fluctuations Enables Fold Adaptability and Specificity.

Recent studies have drawn attention to the evolution of protein dynamics, in addition to sequence and structure, based on the premise structure-encodes-dynamics-encodes-function. Of interest is to understand how functional differentiation is accomplished while maintaining the fold, or how intrinsic dynamics plays out in the evolution of structural variations and functional specificity. We performed a systematic computational analysis of 26,899 proteins belonging to 116 CATH superfamilies. Characterizing cooperative mechanisms and convergent/divergent features that underlie the shared/differentiated dynamics of family members required a methodology that lends itself to efficient analyses of large ensembles of proteins. We therefore introduced, SignDy, an integrated pipeline for evaluating the signature dynamics of families based on elastic network models. Our analysis confirmed that family members share conserved, highly cooperative (global) modes of motion. Importantly, our analysis discloses a subset of motions that sharply distinguishes subfamilies, which lie in a low-to-intermediate frequency regime of the mode spectrum. This regime has maximal impact on functional differentiation of families into subfamilies, while being evolutionarily conserved among subfamily members. Notably, the high-frequency end of the spectrum also reveals evolutionary conserved features across and within subfamilies; but in sharp contrast to global motions, high-frequency modes are minimally collective. Modulation of robust/conserved global dynamics by low-to-intermediate frequency fluctuations thus emerges as a versatile mechanism ensuring the adaptability of selected folds and the specificity of their subfamilies. SignDy further allows for dynamics-based categorization as a new layer of information relevant to distinctive mechanisms of action of subfamilies, beyond sequence or structural classifications.

Accuracy improvement of electrochemical whole blood ketone sensor based on HCT compensation algorithm.

In this manuscript, we demonstrated an electrochemical test strip with HCT (hematocrit) compensation algorithm to improve the accuracy of blood ketone sensor. In the conventional electrochemical sensor, the electrochemical current was directly resolved into the concentration value of the determinant without HCT compensation. For lower or higher HCT blood sample, the measured result was inaccurate. In the proposed design, the blood impedance can be measured to estimate the HCT, which was utilized to compensate the electrochemical current to resolve the more accurate concentration of determinant. The practical blood sample tests demonstrated the proposed design can provide more believable and reliable measured result in clinical point-of-care setting.

Characterization of Differential Dynamics, Specificity, and Allostery of Lipoxygenase Family Members.

Accurate modeling of structural dynamics of proteins and their differentiation across different species can help us understand generic mechanisms of function shared by family members and the molecular basis of the specificity of individual members. We focused here on the family of lipoxygenases, enzymes that catalyze lipid oxidation, the mammalian and bacterial structures of which have been elucidated. We present a systematic method of approach for characterizing the sequence, structure, dynamics, and allosteric signaling properties of these enzymes using a combination of structure-based models and methods and bioinformatics tools applied to a data set of 88 structures. The analysis elucidates the signature dynamics of the lipoxygenase family and its differentiation among members, as well as key sites that enable its adaptation to specific substrate binding and allosteric activity.

Application of MALDI-TOF MS to rapid identification of anaerobic bacteria.

BACKGROUND: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been rapidly developed and widely used as an analytical technique in clinical laboratories with high accuracy in microorganism identification. OBJECTIVE: To validate the efficacy of MALDI-TOF MS in identification of clinical pathogenic anaerobes. METHODS: Twenty-eight studies covering 6685 strains of anaerobic bacteria were included in this meta-analysis. Fixed-effects models based on the P-value and the I-squared were used for meta-analysis to consider the possibility of heterogeneity between studies. Statistical analyses were performed by using STATA 12.0. RESULTS: The identification accuracy of MALDI-TOF MS was 84% for species (I2 = 98.0%, P < 0.1), and 92% for genus (I2 = 96.6%, P < 0.1). Thereinto, the identification accuracy of Bacteroides was the highest at 96% with a 95% CI of 95-97%, followed by Lactobacillus spp., Parabacteroides spp., Clostridium spp., Propionibacterium spp., Prevotella spp., Veillonella spp. and Peptostreptococcus spp., and their correct identification rates were all above 90%, while the accuracy of rare anaerobic bacteria was relatively low. Meanwhile, the overall capabilities of two MALDI-TOF MS systems were different. The identification accuracy rate was 90% for VITEK MS vs. 86% for MALDI biotyper system. CONCLUSIONS: Our research showed that MALDI-TOF-MS was satisfactory in genus identification of clinical pathogenic anaerobic bacteria. However, this method still suffers from different drawbacks in precise identification of rare anaerobe and species levels of common anaerobic bacteria.

DynOmics: dynamics of structural proteome and beyond.

DynOmics (dynomics.pitt.edu) is a portal developed to leverage rapidly growing structural proteomics data by efficiently and accurately evaluating the dynamics of structurally resolved systems, from individual molecules to large complexes and assemblies, in the context of their physiological environment. At the core of the portal is a newly developed server, ENM 1.0, which permits users to efficiently generate information on the collective dynamics of any structure in PDB format, user-uploaded or database-retrieved. ENM 1.0 integrates two widely used elastic network models (ENMs)-the Gaussian Network Model (GNM) and the Anisotropic Network Model (ANM), extended to take account of molecular environment. It enables users to assess potentially functional sites, signal transduction or allosteric communication mechanisms, and protein-protein and protein-DNA interaction poses, in addition to delivering ensembles of accessible conformers reconstructed at atomic details based on the global modes of motions predicted by the ANM. The 'environment' is defined in a flexible manner, from lipid bilayer and crystal contacts, to substrate or ligands bound to a protein, or surrounding subunits in a multimeric structure or assembly. User-friendly interactive features permit users to easily visualize how the environment alter the intrinsic dynamics of the query systems. ENM 1.0 can be accessed at http://enm.pitt.edu/ or http://dyn.life.nthu.edu.tw/oENM/.

Decreased intracellular chloride promotes ADP induced platelet activation through inhibition of cAMP/PKA instead of activation of Lyn/PI3K/Akt pathway.

Decrease of chloride concentration contributes to cardiovascular diseases, however, whether decrease of chloride concentration is involved in platelet activation remains elusive. In the present study, we found that ACI patients had lower serum chloride which would be rescued after Aspirin administration. ADP induced chloride concentration reduction in platelets. Blockade of chloride channel prevented ADP-induced platelet adhesion, activation and aggregation, however, decreasing the extracellular chloride concentration promoted ADP-induced platelet adhesion and activation. Decrease of the extracellular chloride concentration facilitated the inactivation of Src family kinase Lyn, which was not involved in PI3K/Akt phosphorylation. Nevertheless, low chloride concentration promoted the production of platelet cytosol Gαi2 subunit. This subunit prevents AC from converting ATP into cAMP, which therefore, inhibited the phosphorylation of PKA to promote platelet activation. In conclusion, decreased intracellular chloride promotes ADP induced platelet activation through the Gαi2/cAMP/PKA pathway instead of the Lyn/PI3K/Akt signal pathway.

Recent advances in rapid pathogen detection method based on biosensors.

As strain variation and drug resistance become more pervasive, the prevention and control of infection have been a serious problem in recent years. The detection of pathogen is one of the most important parts of the process of diagnosis. Having a series of advantages, such as rapid response, high sensitivity, ease of use, and low cost, biosensors have received much attention and been studied deeply. Moreover, relying on its characteristics of small size, real time, and multiple analyses, biosensors have developed rapidly and used widely and are expected to be applied for microbiological detection in order to meet higher accuracy required by clinical diagnosis. The main goal of this contribution is not to simply collect and list all papers related to pathogen detection based on biosensors published recently, but to discuss critically the development and application of many kinds of biosensors such as electrochemical (amperometric, impedimetric, potentiometric, and conductometric), optical (fluorescent, fibre optic and surface plasmon resonance), and piezoelectric (quartz crystal microbalances and atomic force microscopy) biosensors in pathogen detection as well as the comparisons with the existing clinical detection methods (traditional culture, enzyme-linked immunosorbent assay, polymerase chain reaction, and mass spectrometry).

Aggravated intestinal apoptosis by ClC-3 deletion is lethal to mice endotoxemia.

Our previous study found that ClC-3 chloride channel functioned differently in the vascular and intestinal inflammation, the loss of ClC-3 reduced vascular inflammation but exacerbated intestinal inflammation. To furtherly clarify the role of ClC-3 chloride channels in systemic inflammation, we used LPS-induced endotoxemia model to investigate the response of wild-type and ClC-3 knockout mice to systemic inflammation. The results showed that in the LPS-induced endotoxemia model, the mortality of mice with ClC-3 deletion was significantly higher than that of wild-type mice. The liver and lung inflammations in mice with ClC-3 deletion were significantly less than those in wild-type mice, and the levels of TNF-α and MIP-2 in serum were lower than those of wild-type mice. However, intestinal inflammatory cytokines contents and intestinal permeability were higher than wild-type mice. After transfection of THP-1 cells with ClC-3 siRNA, the contents of TNF-α and IL-8 in LPS-induced cell supernatants were significantly decreased. Further experiments revealed that the level of Bax and Cleaved Caspase 3 in intestinal tissue of mice with ClC-3 deletion was significantly increased, while the level of Bcl2 did not change, which indicated that the intestinal apoptosis was increased after LPS-induced mice intestinal integrity destruction. Therefore, the regulation of intestinal tissue integrity by ClC-3 is crucial for maintaining LPS-induced survival in mice with endotoxemia.