In pursuit of resolution in time-of-flight mass spectrometry: A historical perspective.

Time-of-flight mass spectrometry is reviewed from its inception in the 1940s to the present day. The review is concerned with fundamentals of time-of-flight analyzers and of ion sources to the extent that sources influence analyzers. The patent literature has been covered, and efforts made to bring to light less well-known papers and studies © 2015 Wiley Periodicals, Inc. Mass Spec Rev. 35:738-757, 2016.

Using the gini coefficient to measure the chemical diversity of small-molecule libraries.

Modern databases of small organic molecules contain tens of millions of structures. The size of theoretically available chemistry is even larger. However, despite the large amount of chemical information, the "big data" moment for chemistry has not yet provided the corresponding payoff of cheaper computer-predicted medicine or robust machine-learning models for the determination of efficacy and toxicity. Here, we present a study of the diversity of chemical datasets using a measure that is commonly used in socioeconomic studies. We demonstrate the use of this diversity measure on several datasets that were constructed to contain various congeneric subsets of molecules as well as randomly selected molecules. We also apply our method to a number of well-known databases that are frequently used for structure-activity relationship modeling. Our results show the poor diversity of the common sources of potential lead compounds compared to actual known drugs. © 2016 Wiley Periodicals, Inc.

Development and implementation of (Q)SAR modeling within the CHARMMing web-user interface.

Recent availability of large publicly accessible databases of chemical compounds and their biological activities (PubChem, ChEMBL) has inspired us to develop a web-based tool for structure activity relationship and quantitative structure activity relationship modeling to add to the services provided by CHARMMing (www.charmming.org). This new module implements some of the most recent advances in modern machine learning algorithms-Random Forest, Support Vector Machine, Stochastic Gradient Descent, Gradient Tree Boosting, so forth. A user can import training data from Pubchem Bioassay data collections directly from our interface or upload his or her own SD files which contain structures and activity information to create new models (either categorical or numerical). A user can then track the model generation process and run models on new data to predict activity.

Cytotoxic CD8+ Temra cells show loss of chromatin accessibility at genes associated with T cell activation.

As humans age, their memory T cell compartment expands due to the lifelong exposure to antigens. This expansion is characterized by terminally differentiated CD8+ T cells (Temra), which possess NK cell-like phenotype and are associated with chronic inflammatory conditions. Temra cells are predominantly driven by the sporadic reactivation of cytomegalovirus (CMV), yet their epigenomic patterns and cellular heterogeneity remain understudied. To address this gap, we correlated their gene expression profiles with chromatin openness and conducted single-cell transcriptome analysis, comparing them to other CD8+ subsets and CMV-responses. We confirmed that Temra cells exhibit high expression of genes associated with cytotoxicity and lower expression of costimulatory and chemokine genes. The data revealed that CMV-responsive CD8+ T cells (Tcmv) were predominantly derived from a mixed population of Temra and memory cells (Tcm/em) and shared their transcriptomic profiles. Using ATAC-seq analysis, we identified 1449 differentially accessible chromatin regions between CD8+ Temra and Tcm/em cells, of which only 127 sites gained chromatin accessibility in Temra cells. We further identified 51 gene loci, including costimulatory CD27, CD28, and ICOS genes, whose chromatin accessibility correlated with their gene expression. The differential chromatin regions Tcm/em cells were enriched in motifs that bind multiple transcriptional activators, such as Jun/Fos, NFkappaB, and STAT, whereas the open regions in Temra cells mainly contained binding sites of T-box transcription factors. Our single-cell analysis of CD8+CCR7loCD45RAhi sorted Temra population showed several subsets of Temra and NKT-like cells and CMC1+ Temra populations in older individuals that were shifted towards decreased cytotoxicity. Among CD8+CCR7loCD45RAhi sorted cells, we found a decreased proportion of IL7R+ Tcm/em-like and MAIT cells in individuals with high levels of CMV antibodies (CMVhi). These results shed new light on the molecular and cellular heterogeneity of CD8+ Temra cells and their relationship to aging and CMV infection.

Single cell characterization of blood and expanded regulatory T cells in autoimmune polyendocrine syndrome type 1.

Immune tolerance fails in autoimmune polyendocrine syndrome type 1 (APS-1) because of AIRE mutations. We have used single cell transcriptomics to characterize regulatory T cells (Tregs) sorted directly from blood and from in vitro expanded Tregs in APS-1 patients compared to healthy controls. We revealed only CD52 and LTB (down) and TXNIP (up) as consistently differentially expressed genes in the datasets. There were furthermore no large differences of the TCR-repertoire of expanded Tregs between the cohorts, but unique patients showed a more restricted use of specific clonotypes. We also found that in vitro expanded Tregs from APS-1 patients had similar suppressive capacity as controls in co-culture assays, despite expanding faster and having more exhausted cells. Our results suggest that APS-1 patients do not have intrinsic defects in their Treg functionality, and that their Tregs can be expanded ex vivo for potential therapeutic applications.

Inhibitors for the hepatitis C virus RNA polymerase explored by SAR with advanced machine learning methods.

Hepatitis C virus (HCV) is a global health challenge, affecting approximately 200 million people worldwide. In this study we developed SAR models with advanced machine learning classifiers Random Forest and k Nearest Neighbor Simulated Annealing for 679 small molecules with measured inhibition activity for NS5B genotype 1b. The activity was expressed as a binary value (active/inactive), where actives were considered molecules with IC50 ≤0.95 µM. We applied our SAR models to various drug-like databases and identified novel chemical scaffolds for NS5B inhibitors. Subsequent in vitro antiviral assays suggested a new activity for an existing prodrug, Candesartan cilexetil, which is currently used to treat hypertension and heart failure but has not been previously tested for anti-HCV activity. We also identified NS5B inhibitors with two novel non-nucleoside chemical motifs.

Nuclear inelastic scattering and Mössbauer spectroscopy as local probes for ligand binding modes and electronic properties in proteins: vibrational behavior of a ferriheme center inside a ß-barrel protein.

In this work, we present a study of the influence of the protein matrix on its ability to tune the binding of small ligands such as NO, cyanide (CN(-)), and histamine to the ferric heme iron center in the NO-storage and -transport protein Nitrophorin 2 (NP2) from the salivary glands of the blood-sucking insect Rhodnius prolixus. Conventional Mössbauer spectroscopy shows a diamagnetic ground state of the NP2-NO complex and Type I and II electronic ground states of the NP2-CN(-) and NP2-histamine complex, respectively. The change in the vibrational signature of the protein upon ligand binding has been monitored by Nuclear Inelastic Scattering (NIS), also called Nuclear Resonant Vibrational Spectroscopy (NRVS). The NIS data thus obtained have also been calculated by quantum mechanical (QM) density functional theory (DFT) coupled with molecular mechanics (MM) methods. The calculations presented here show that the heme ruffling in NP2 is a consequence of the interaction with the protein matrix. Structure optimizations of the heme and its ligands with DFT retain the characteristic saddling and ruffling only if the protein matrix is taken into account. Furthermore, simulations of the NIS data by QM/MM calculations suggest that the pH dependence of the binding of NO, but not of CN(-) and histamine, might be a consequence of the protonation state of the heme carboxyls.

PDB ligand conformational energies calculated quantum-mechanically.

We present here a greatly updated version of an earlier study on the conformational energies of protein-ligand complexes in the Protein Data Bank (PDB) [Nicklaus et al. Bioorg. Med. Chem. 1995, 3, 411-428], with the goal of improving on all possible aspects such as number and selection of ligand instances, energy calculations performed, and additional analyses conducted. Starting from about 357,000 ligand instances deposited in the 2008 version of the Ligand Expo database of the experimental 3D coordinates of all small-molecule instances in the PDB, we created a "high-quality" subset of ligand instances by various filtering steps including application of crystallographic quality criteria and structural unambiguousness. Submission of 640 Gaussian 03 jobs yielded a set of about 415 successfully concluded runs. We used a stepwise optimization of internal degrees of freedom at the DFT level of theory with the B3LYP/6-31G(d) basis set and a single-point energy calculation at B3LYP/6-311++G(3df,2p) after each round of (partial) optimization to separate energy changes due to bond length stretches vs bond angle changes vs torsion changes. Even for the most "conservative" choice of all the possible conformational energies-the energy difference between the conformation in which all internal degrees of freedom except torsions have been optimized and the fully optimized conformer-significant energy values were found. The range of 0 to ~25 kcal/mol was populated quite evenly and independently of the crystallographic resolution. A smaller number of "outliers" of yet higher energies were seen only at resolutions above 1.3 Å. The energies showed some correlation with molecular size and flexibility but not with crystallographic quality metrics such as the Cruickshank diffraction-component precision index (DPI) and R(free)-R, or with the ligand instance-specific metrics such as occupancy-weighted B-factor (OWAB), real-space R factor (RSR), and real-space correlation coefficient (RSCC). We repeated these calculations with the solvent model IEFPCM, which yielded energy differences that were generally somewhat lower than the corresponding vacuum results but did not produce a qualitatively different picture. Torsional sampling around the crystal conformation at the molecular mechanics level using the MMFF94s force field typically led to an increase in energy.

Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability.

Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in "fixed" relationships, either antiperiplanar where the AE is permanently "on", or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values.

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding.

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.

Dynamics of infraslow potentials in the primary auditory cortex: component analysis and contribution of specific thalamic-cortical and non-specific brainstem-cortical influences.

Several available reports demonstrate the presence of infraslow activity (<0.5 Hz) in structures of the auditory system of the brain. It was reported earlier that specific alterations of this activity in the domain of seconds (0.1-0.5 Hz) occurred in the medial geniculate nucleus (MGN) and primary auditory cortex (A1) in response to acoustic stimuli. The present study was performed to test two hypotheses: (1) that potentials in the domain of seconds (0.1-0.5 Hz) reflect specific and direct interactions of the MGN and A1 during neural processing of sensory information, and (2) that low-frequency infraslow potentials in the A1 (<0.1 Hz) are related to brainstem influences originating from the locus coeruleus (LC) and dorsal raphe nucleus (DRN). The experimental subjects were 25 adult rats with chronic stereotaxic electrodes implanted in the MGN, A1, LC, and DRN. The animals were anesthetized and infraslow activity was once recorded under several experimental conditions: (1) in the A1 before and after electrical stimulation of MGN, (2) in the A1 before and after electrical stimulation of LC, and (3) in the A1 before and after electrical stimulation of DRN. The effects of MGN stimulation were limited to overall increases in spectral power in the frequency domain of 0.1-0.5 Hz. Specifically, power increased in the frequencies of 0.1-0.25, 0.35-0.4, and 0.45-0.5 Hz in the A1 after MGN stimulation. The electrical stimulation of either the LC or DRN affected only multisecond activity (0.0167-0.04 Hz) in the A1 in the similar way (increase of powers of multisecond potentials), but it does not induced any changes in the activity with the frequencies of 0.1-0.5 Hz in this structure. These results support tentative conclusions that infraslow activity in the range of 0.1-0.5 Hz is implicated in specific mechanisms of interactions within the MGN-A1 thalamic-cortical system, whereas multisecond potentials (0.0167-0.04 Hz) in A1 are mainly attributed to the influences of brainstem nuclei (like LC and DRN) on general neuronal excitability of this auditory cortical area.

Sound-induced changes of infraslow brain potential fluctuations in the medial geniculate nucleus and primary auditory cortex in anaesthetized rats.

Recent publications indicate the presence of infraslow activity (<0.5 Hz) in subcortical and cortical sites of the auditory system of the brain. It has been reported that this activity might be sensitive to acoustic stimuli. Yet the dynamics of infraslow brain potential (ISBP) fluctuations in these structures and their potential sensitivity to auditory stimuli are unknown. The present study was performed in order to test the hypothesis that extracellular ISBP activity in the medial geniculate nucleus (MGN) and the primary auditory cortex (A1) responds concurrently to acoustic stimuli. The experimental subjects were 5 adult rats with chronic stereotaxic electrodes implanted in MGN and A1. The animals were anesthetized and recordings were made in both sites during both silence and rhythmical acoustic stimulation. Our results support the hypothesis that these fluctuations are sensitive to acoustic stimuli. There were similar changes in ISBP activity in the MGN and A1 in response to rhythmic acoustic stimulation. Specifically, there were significant increases in the frequency range of seconds. Based on these findings, we suggest that sound-correlated changes in infraslow activity in the range of seconds in the MGN and A1 reflect specific mechanisms of neural processing of acoustic information in the auditory system of the brain.

Very slow brain potential fluctuations (< 0.5 Hz) in visual thalamus and striate cortex after their successive electrical stimulation in lightly anesthetized rats.

Recently accumulating evidence demonstrates the presence of very slow activity (< 0.5 Hz) in structures of the visual system of the brain. It was found in our laboratory earlier that specific and significant alterations of this activity (mainly in the domain of seconds) occurred in the visual system in response to illumination changes. The present study was performed in order to test the hypothesis that potentials in the domain of seconds reflect specific and direct interactions of the lateral geniculate nucleus (LGN) and the primary visual cortex (V1) during neural processing of sensory information. The experimental subjects were seven adult rats with chronic stereotaxic electrodes implanted in the LGN and V1. Animals were lightly anesthetized and the recordings were made in the LGN before and after electrical stimulation of V1 and also in the V1 before and after stimulation of LGN. The main findings were significant spectral changes in the domain of seconds in the V1 after LGN electrical stimulation and similar changes in the LGN after V1 electrical stimulation. These changes were manifested as significant increases in power in the domain of seconds (0.1-0.5 Hz). Significant responses were detected in the both LGN and V1 multisecond activities (pre- vs. post-stimulus recordings). The changes were opposite in direction in the LGN and V1. The obtained results support the conclusion that very slow activity in the domain of seconds reflects specific mechanisms of forward and backward interactions within the LGN-V1 thalamic-cortical-thalamic system, while multisecond activity relates to global neuronal activity fluctuations.

Very slow potentials in the lateral geniculate complex and primary visual cortex during different illumination changes in freely moving rats.

Previous literature has shown different forms of very slow oscillatory phenomena (0-0.5 Hz) in the structures of brain visual system. It was demonstrated in aforementioned publications that this infraslow activity might be sensitive to the level of environmental illumination. This study was performed to test the hypothesis that extracellular very slow brain potential (VSBP) oscillations or fluctuations in lateral geniculate complex (LGC) and primary visual cortex (PVC) are responding specifically and concurrently to different illumination changes. Experiments were conducted on five albino rats with chronically implanted stereotaxic electrodes in LGC and PVC brain sites. Our results support the aforementioned suggestion and revealed significant and similar patterns of VSBP modification within a frequency domain of seconds (0.1-0.25 Hz) in both the LGC and PVC in response to darkness, illumination and photostimulation. It is also documented here that significant and similar changes of multisecond activity (0.02-0.04 Hz) occur in both investigated brain sites in response to photostimulation only. Based on these findings, we propose that a possible role for VSBP oscillations in the LGC and PVC should be strongly considered in the CNS mechanisms of visual information processing.

Very slow potential oscillations in locus coeruleus and dorsal raphe nucleus under different illumination in freely moving rats.

Recent findings have revealed very slow (<0.5 Hz) oscillatory phenomena in the structures of the brain visual system. It has been proposed that very slow brain potentials in an extremely slow domain, less than 0.1 Hz, recorded from the lateral geniculate complex and primary visual cortex are associated with periodic influences originating from the locus coeruleus and dorsal raphe nucleus. The present study was performed to test the hypothesis that extremely slow brain potential oscillatory patterns in the locus coeruleus and dorsal raphe nucleus during several types of visual stimulation--light exposure, darkness, and photostimulation--are similar to those in the primary visual cortex and lateral geniculate complex under the same conditions of illumination. The results support this hypothesis. Specifically, spectral patterns of multisecond oscillations in the range of 0.02-0.04 Hz and fluctuations in the domain of minutes (below 0.002 Hz) were present in both the locus coeruleus and dorsal raphe nucleus and were similar to those found in the primary visual cortex and lateral geniculate complex. Additionally, we detected significant increases in the power spectra of multisecond oscillations in both nuclei in response to photostimulation (P<0.05). Our tentative conclusion is that extremely slow potentials in the locus coeruleus and dorsal raphe nucleus contribute to the regulation of extremely slow activity in the brain visual system.

Genetic deletion of transglutaminase 2 does not rescue the phenotypic deficits observed in R6/2 and zQ175 mouse models of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by expansion of CAG repeats in the huntingtin gene. Tissue transglutaminase 2 (TG2), a multi-functional enzyme, was found to be increased both in HD patients and in mouse models of the disease. Furthermore, beneficial effects have been reported from the genetic ablation of TG2 in R6/2 and R6/1 mouse lines. To further evaluate the validity of this target for the treatment of HD, we examined the effects of TG2 deletion in two genetic mouse models of HD: R6/2 CAG 240 and zQ175 knock in (KI). Contrary to previous reports, under rigorous experimental conditions we found that TG2 ablation had no effect on either motor or cognitive deficits, or on the weight loss. In addition, under optimal husbandry conditions, TG2 ablation did not extend R6/2 lifespan. Moreover, TG2 deletion did not change the huntingtin aggregate load in cortex or striatum and did not decrease the brain atrophy observed in either mouse line. Finally, no amelioration of the dysregulation of striatal and cortical gene markers was detected. We conclude that TG2 is not a valid therapeutic target for the treatment of HD.

High-Throughput Automated Phenotyping of Two Genetic Mouse Models of Huntington's Disease.

Phenotyping with traditional behavioral assays constitutes a major bottleneck in the primary screening, characterization, and validation of genetic mouse models of disease, leading to downstream delays in drug discovery efforts. We present a novel and comprehensive one-stop approach to phenotyping, the PhenoCube™. This system simultaneously captures the cognitive performance, motor activity, and circadian patterns of group-housed mice by use of home-cage operant conditioning modules (IntelliCage) and custom-built computer vision software. We evaluated two different mouse models of Huntington's Disease (HD), the R6/2 and the BACHD in the PhenoCube™ system. Our results demonstrated that this system can efficiently capture and track alterations in both cognitive performance and locomotor activity patterns associated with these disease models. This work extends our prior demonstration that PhenoCube™ can characterize circadian dysfunction in BACHD mice and shows that this system, with the experimental protocols used, is a sensitive and efficient tool for a first pass high-throughput screening of mouse disease models in general and mouse models of neurodegeneration in particular.

Computational tools and resources for metabolism-related property predictions. 2. Application to prediction of half-life time in human liver microsomes.

BACKGROUND: The most important factor affecting metabolic excretion of compounds from the body is their half-life time. This provides an indication of compound stability of, for example, drug molecules. We report on our efforts to develop QSAR models for metabolic stability of compounds, based on in vitro half-life assay data measured in human liver microsomes. METHOD: A variety of QSAR models generated using different statistical methods and descriptor sets implemented in both open-source and commercial programs (KNIME, GUSAR and StarDrop) were analyzed. The models obtained were compared using four different external validation sets from public and commercial data sources, including two smaller sets of in vivo half-life data in humans. CONCLUSION: In many cases, the accuracy of prediction achieved on one external test set did not correspond to the results achieved with another test set. The most predictive models were used for predicting the metabolic stability of compounds from the open NCI database, the results of which are publicly available on the NCI/CADD Group web server ( http://cactus.nci.nih.gov ).

Rapid, computer vision-enabled murine screening system identifies neuropharmacological potential of two new mechanisms.

The lack of predictive in vitro models for behavioral phenotypes impedes rapid advancement in neuropharmacology and psychopharmacology. In vivo behavioral assays are more predictive of activity in human disorders, but such assays are often highly resource-intensive. Here we describe the successful application of a computer vision-enabled system to identify potential neuropharmacological activity of two new mechanisms. The analytical system was trained using multiple drugs that are used clinically to treat depression, schizophrenia, anxiety, and other psychiatric or behavioral disorders. During blinded testing the PDE10 inhibitor TP-10 produced a signature of activity suggesting potential antipsychotic activity. This finding is consistent with TP-10's activity in multiple rodent models that is similar to that of clinically used antipsychotic drugs. The CK1ε inhibitor PF-670462 produced a signature consistent with anxiolytic activity and, at the highest dose tested, behavioral effects similar to that of opiate analgesics. Neither TP-10 nor PF-670462 was included in the training set. Thus, computer vision-based behavioral analysis can facilitate drug discovery by identifying neuropharmacological effects of compounds acting through new mechanisms.

Circadian Abnormalities in Motor Activity in a BAC Transgenic Mouse Model of Huntington's Disease.

Huntington's disease (HD) is a progressive neurodegenerative disease marked by psychiatric and motor problems. Recently, these findings have been extended to deficits in sleep and circadian function that can be observed in HD patients and in HD mouse models, with abnormal sleep patterns correlating with symptom severity in patients. Here, we studied the behavior of the BAC HD mouse model using an 24/7 automated system; the results indicate significant lengthening of the circadian period in the mutant mice. These results reinforce previous findings in HD models and symptomatic HD patients, indicating that circadian dysfunction is a core feature of HD.