Live Detection of Neural Progenitors and Glioblastoma Cells by an Oligothiophene Derivative.

There is an urgent need for simple and non-invasive identification of live neural stem/progenitor cells (NSPCs) in the developing and adult brain as well as in disease, such as in brain tumors, due to the potential clinical importance in prognosis, diagnosis, and treatment of diseases of the nervous system. Here, we report a luminescent conjugated oligothiophene (LCO), named p-HTMI, for non-invasive and non-amplified real-time detection of live human patient-derived glioblastoma (GBM) stem cell-like cells and NSPCs. While p-HTMI stained only a small fraction of other cell types investigated, the mere addition of p-HTMI to the cell culture resulted in efficient detection of NSPCs or GBM cells from rodents and humans within minutes. p-HTMI is functionalized with a methylated imidazole moiety resembling the side chain of histidine/histamine, and non-methylated analogues were not functional. Cell sorting experiments of human GBM cells demonstrated that p-HTMI labeled the same cell population as CD271, a proposed marker for stem cell-like cells and rapidly migrating cells in glioblastoma. Our results suggest that the LCO p-HTMI is a versatile tool for immediate and selective detection of neural and glioma stem and progenitor cells.

The link between liver fat and cardiometabolic diseases is highlighted by genome-wide association study of MRI-derived measures of body composition.

Obesity and associated morbidities, metabolic associated fatty liver disease (MAFLD) included, constitute some of the largest public health threats worldwide. Body composition and related risk factors are known to be heritable and identification of their genetic determinants may aid in the development of better prevention and treatment strategies. Recently, large-scale whole-body MRI data has become available, providing more specific measures of body composition than anthropometrics such as body mass index. Here, we aimed to elucidate the genetic architecture of body composition, by conducting genome-wide association studies (GWAS) of these MRI-derived measures. We ran both univariate and multivariate GWAS on fourteen MRI-derived measurements of adipose and muscle tissue distribution, derived from scans from 33,588 White European UK Biobank participants (mean age of 64.5 years, 51.4% female). Through multivariate analysis, we discovered 100 loci with distributed effects across the body composition measures and 241 significant genes primarily involved in immune system functioning. Liver fat stood out, with a highly discoverable and oligogenic architecture and the strongest genetic associations. Comparison with 21 common cardiometabolic traits revealed both shared and specific genetic influences, with higher mean heritability for the MRI measures (h2 = .25 vs. .13, p = 1.8x10-7). We found substantial genetic correlations between the body composition measures and a range of cardiometabolic diseases, with the strongest correlation between liver fat and type 2 diabetes (rg = .49, p = 2.7x10-22). These findings show that MRI-derived body composition measures complement conventional body anthropometrics and other biomarkers of cardiometabolic health, highlighting the central role of liver fat, and improving our knowledge of the genetic architecture of body composition and related diseases.

Irritable bowel syndrome in women: Association between decreased insular subregion volumes and gastrointestinal symptoms.

OBJECTIVE: Irritable bowel syndrome (IBS) is a chronic pain disorder characterized by disturbed interactions between the gut and the brain with depression as a common comorbidity. In both IBS and depression, structural brain alterations of the insular cortices, key structures for pain processing and interoception, have been demonstrated but the specificity of these findings remains unclear. We compared the gray matter volume (GMV) of insular cortex (IC) subregions in IBS women and healthy controls (HC) and examined relations to gastrointestinal (GI) symptoms and glutamate + glutamine (Glx) concentrations. We further analyzed GMV of IC subregions in women with major depression (MDD) compared to HC and addressed possible differences between depression, IBS, IBS with depression and HC. DESIGN: Women with IBS (n = 75), MDD (n = 41) and their respective HC (n = 39 and n = 43) underwent structural brain MRI. IC subregion volumes were estimated using statistical parametric mapping software. General linear model approaches were applied to IC volumetric data and FDR-corrected partial correlation analyses assessed relations between GMV, GI symptoms and Glx concentrations. RESULTS: IBS patients had significantly smaller IC subregions than HC in both hemispheres but there was no significant difference between MDD compared with IBS and HC for any insular subregion. In IBS, the dorsal anterior insular volumes were negatively correlated with symptoms of nausea and pain, and the left ventral subregion showed a positive correlation with straining to defecate, while the posterior subregion volumes showed no relation to symptoms. In the anterior insula, concentration of Glx showed positive correlations with GMV bilaterally in HC and with GMV of the right anterior insula in IBS. CONCLUSION: As the interoceptive cortex, the insula shows substantial and disease-specific structural differences in patients with chronic interoceptive visceral pain. Particularly changes in the anterior proportions might be related to chronic exposure to or enhanced salience towards adverse interoceptive visceral signals and could be linked to biochemical changes, calling for further multimodal and longitudinal work.

Adipose tissue distribution from body MRI is associated with cross-sectional and longitudinal brain age in adults.

There is an intimate body-brain connection in ageing, and obesity is a key risk factor for poor cardiometabolic health and neurodegenerative conditions. Although research has demonstrated deleterious effects of obesity on brain structure and function, the majority of studies have used conventional measures such as waist-to-hip ratio, waist circumference, and body mass index. While sensitive to gross features of body composition, such global anthropometric features fail to describe regional differences in body fat distribution and composition. The sample consisted of baseline brain magnetic resonance imaging (MRI) acquired from 790 healthy participants aged 18-94 years (mean ± standard deviation (SD) at baseline: 46.8 ± 16.3), and follow-up brain MRI collected from 272 of those individuals (two time-points with 19.7 months interval, on average (min = 9.8, max = 35.6). Of the 790 included participants, cross-sectional body MRI data was available from a subgroup of 286 participants, with age range 19-86 (mean = 57.6, SD = 15.6). Adopting a mixed cross-sectional and longitudinal design, we investigated cross-sectional body magnetic resonance imaging measures of adipose tissue distribution in relation to longitudinal brain structure using MRI-based morphometry (T1) and diffusion tensor imaging (DTI). We estimated tissue-specific brain age at two time points and performed Bayesian multilevel modelling to investigate the associations between adipose measures at follow-up and brain age gap (BAG) - the difference between actual age and the prediction of the brain's biological age - at baseline and follow-up. We also tested for interactions between BAG and both time and age on each adipose measure. The results showed credible associations between T1-based BAG and liver fat, muscle fat infiltration (MFI), and weight-to-muscle ratio (WMR), indicating older-appearing brains in people with higher measures of adipose tissue. Longitudinal evidence supported interaction effects between time and MFI and WMR on T1-based BAG, indicating accelerated ageing over the course of the study period in people with higher measures of adipose tissue. The results show that specific measures of fat distribution are associated with brain ageing and that different compartments of adipose tissue may be differentially linked with increased brain ageing, with potential to identify key processes involved in age-related transdiagnostic disease processes.

Population-based body-brain mapping links brain morphology with anthropometrics and body composition.

Understanding complex body-brain processes and the interplay between adipose tissue and brain health is important for understanding comorbidity between psychiatric and cardiometabolic disorders. We investigated associations between brain structure and anthropometric and body composition measures using brain magnetic resonance imaging (MRI; n = 24,728) and body MRI (n = 4973) of generally healthy participants in the UK Biobank. We derived regional and global measures of brain morphometry using FreeSurfer and tested their association with (i) anthropometric measures, and (ii) adipose and muscle tissue measured from body MRI. We identified several significant associations with small effect sizes. Anthropometric measures showed negative, nonlinear, associations with cerebellar/cortical gray matter, and brain stem structures, and positive associations with ventricular volumes. Subcortical structures exhibited mixed effect directionality, with strongest positive association for accumbens. Adipose tissue measures, including liver fat and muscle fat infiltration, were negatively associated with cortical/cerebellum structures, while total thigh muscle volume was positively associated with brain stem and accumbens. Regional investigations of cortical area, thickness, and volume indicated widespread and largely negative associations with anthropometric and adipose tissue measures, with an opposite pattern for thigh muscle volume. Self-reported diabetes, hypertension, or hypercholesterolemia were associated with brain structure. The findings provide new insight into physiological body-brain associations suggestive of shared mechanisms between cardiometabolic risk factors and brain health. Whereas the causality needs to be determined, the observed patterns of body-brain relationships provide a foundation for understanding the underlying mechanisms linking psychiatric disorders with obesity and cardiovascular disease, with potential for the development of new prevention strategies.

Vasoactive intestinal polypeptide plasma levels associated with affective symptoms and brain structure and function in healthy females.

Vasoactive intestinal polypeptide (VIP) is a neuroendocrine peptide distributed throughout the human body, including the CNS, where it is particularly abundant in brain regions associated with anxiety and depression. Based on earlier studies indicating that peripheral VIP may cross through the blood-brain barrier, we hypothesized plasma VIP levels to be associated with symptoms of anxiety and depression, as well as brain volume and resting-state functional connectivity in the amygdala, hippocampus, parahippocampus, and orbitofrontal cortex. Plasma VIP concentrations and anxiety/depression symptoms were measured in 37 healthy females. Functional and structural magnetic resonance imaging were used to evaluate functional connectivity and brain volume respectively, and their associations with VIP concentrations within brain regions associated with anxiety and depression. Negative correlations were found between VIP levels and symptoms of anxiety (r = - 0.44, p = 0.002) and depression (r = - 0.50, p = 0.001). Functional connectivity demonstrated significant VIP-dependent positive associations between the amygdala seed region with both the right parahippocampus (t(33) = 3.1, pFDR = 0.02) and right lateral orbitofrontal cortex (OFC; t(33) = 2.9, pFDR = 0.02). Moreover, VIP concentrations were significantly, positively correlated with brain volume in the left amygdala (r = 0.28, p = 0.007) and left lateral OFC (r = 0.29, p = 0.004). The present findings highlight a potential role for VIP in the neurobiology of affective symptoms.

Fatigue in irritable bowel syndrome is associated with plasma levels of TNF-α and mesocorticolimbic connectivity.

Irritable bowel syndrome (IBS) is a symptom-based disorder of gut-brain interactions generating abdominal pain. It is also associated with a vulnerability to develop extraintestinal symptoms, with fatigue often reported as one of the most disturbing. Fatigue is related to brain function and inflammation in several disorders, however, the mechanisms of such relations in IBS remain elusive. This study aimed to elucidate fatigue and its association with a resting state network of mesocorticolimbic regions of known importance in fatigue, and to explore the possible role of circulating TNF-α levels in IBS and healthy controls (HC). Resting state functional magnetic resonance imaging (fMRI) was conducted in 88 IBS patients and 47 HC of similar age and gender to investigate functional connectivity between mesocorticolimbic regions. Further, fatigue impact on daily life and plasma levels of the proinflammatory cytokine tumor necrosis factor-α (TNF-α), of known relevance to immune activation in IBS, were also measured. The selected mesocorticolimbic regions indeed formed a functionally connected network in all participants. The nucleus accumbens (NAc), in particular, exhibited functional connectivity to all other regions of interest. In IBS, fatigue impact on daily life was negatively correlated with the connectivity between NAc and dorsolateral prefrontal cortex bilaterally (left p = 0.019; right p = 0.038, corrected for multiple comparisons), while in HC, fatigue impact on daily life was positively correlated to the connectivity between the right NAc and anterior middle insula in both hemispheres (left p = 0.009; right p = 0.011). We found significantly higher levels of TNF-α in IBS patients compared to HC (p = 0.001) as well as a positive correlation between TNF-α and fatigue impact on daily life in IBS patients (rho = 0.25, p = 0.02) but not in HC (rho = -0.13, p = 0.37). There was no association between functional connectivity in the mesocorticolimbic network and plasma levels of TNF-α in either group In summary, this novel multimodal study provides the first evidence that the vulnerability to fatigue in IBS is associated with connectivity within a mesocorticolimbic network as well as immune activation. These findings warrant further investigation, both peripherally and potentially with measurements of central immune activation as well.

Visual Analysis for Understanding Irritable Bowel Syndrome.

The cause of irritable bowel syndrome (IBS), a chronic disorder characterized by abdominal pain and disturbed bowel habits, is largely unknown. It is believed to be related to physical properties in the gut, central mechanisms in the brain, psychological factors, or a combination of these. To understand the relationships within the gut-brain axis with respect to IBS, large numbers of measurements ranging from stool samples to functional magnetic resonance imaging are collected from patients with IBS and healthy controls. As such, IBS is a typical example in medical research where research turns into a big data analysis challenge. In this chapter we demonstrate the power of interactive visual data analysis and exploration to generate an environment for scientific reasoning and hypothesis formulation for data from multiple sources with different character. Three case studies are presented to show the utility of the presented work.

Positive Allosteric Modulator of GABA Lowers BOLD Responses in the Cingulate Cortex.

Knowledge about the neural underpinnings of the negative blood oxygen level dependent (BOLD) responses in functional magnetic resonance imaging (fMRI) is still limited. We hypothesized that pharmacological GABAergic modulation attenuates BOLD responses, and that blood concentrations of a positive allosteric modulator of GABA correlate inversely with BOLD responses in the cingulate cortex. We investigated whether or not pure task-related negative BOLD responses were co-localized with pharmacologically modulated BOLD responses. Twenty healthy adults received either 5 mg diazepam or placebo in a double blind, randomized design. During fMRI the subjects performed a working memory task. Results showed that BOLD responses in the cingulate cortex were inversely correlated with diazepam blood concentrations; that is, the higher the blood diazepam concentration, the lower the BOLD response. This inverse correlation was most pronounced in the pregenual anterior cingulate cortex and the anterior mid-cingulate cortex. For subjects with diazepam plasma concentration > 0.1 mg/L we observed negative BOLD responses with respect to fixation baseline. There was minor overlap between cingulate regions with task-related negative BOLD responses and regions where the BOLD responses were inversely correlated with diazepam concentration. We interpret that the inverse correlation between the BOLD response and diazepam was caused by GABA-related neural inhibition. Thus, this study supports the hypothesis that GABA attenuates BOLD responses in fMRI. The minimal overlap between task-related negative BOLD responses and responses attenuated by diazepam suggests that these responses might be caused by different mechanisms.

The default mode network as a biomarker for monitoring the therapeutic effects of meditation.

The default mode network (DMN) is a group of anatomically separate regions in the brain found to have synchronized patterns of activation in functional magnetic resonance imaging (fMRI). Mentation associated with the DMN includes processes such as mind wandering, autobiographical memory, self-reflective thought, envisioning the future, and considering the perspective of others. Abnormalities in the DMN have been linked to symptom severity in a variety of mental disorders indicating that the DMN could be used as a biomarker for diagnosis. These correlations have also led to the use of DMN modulation as a biomarker for assessing pharmacological treatments. Concurrent research investigating the neural correlates of meditation, have associated DMN modulation with practice. Furthermore, meditative practice is increasingly understood to have a beneficial role in the treatment of mental disorders. Therefore we propose the use of DMN measures as a biomarker for monitoring the therapeutic effects of meditation practices in mental disorders. Recent findings support this perspective, and indicate the utility of DMN monitoring in understanding and developing meditative treatments for these debilitating conditions.

An azide functionalized oligothiophene ligand--a versatile tool for multimodal detection of disease associated protein aggregates.

Ligands for identifying protein aggregates are of great interest as such deposits are the pathological hallmark of a wide range of severe diseases including Alzheimer's and Parkinson's disease. Here we report the synthesis of an azide functionalized fluorescent pentameric oligothiophene that can be utilized as a ligand for multimodal detection of disease-associated protein aggregates. The azide functionalization allows for attachment of the ligand to a surface by conventional click chemistry without disturbing selective interaction with protein aggregates and the oligothiophene-aggregate interaction can be detected by fluorescence or surface plasmon resonance. In addition, a methodology where the oligothiophene ligand is employed as a capturing molecule selective for aggregated proteins in combination with an antibody detecting a distinct peptide/protein is also presented. We foresee that this methodology will offer the possibility to create a variety of multiplex sensing systems for sensitive and selective detection of protein aggregates, the pathological hallmarks of several neurodegenerative diseases.

Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands.

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.

Toward a molecular understanding of the detection of amyloid proteins with flexible conjugated oligothiophenes.

Molecular and electronic structures and optical absorption properties of oligothiophenes used for spectral assignment of amyloid deposits have been investigated for a family of probes known as luminescent conjugated oligothiophenes (LCOs). Theoretical absorption spectra have been determined using conformational averaging, combining classical molecular dynamics (MD) simulations with quantum mechanical/molecular mechanics (QM/MM) time-dependent density functional theory (TD-DFT) spectrum calculations. Theoretical absorption spectra are in excellent agreement with experiments, showing average errors below 5 nm for absorption maxima. To couple observed properties to molecular structures, a measure of planarity is defined, revealing a strong correlation between the transition wavelength of the first and dominating electronically excited state and dihedral rotations. It is shown that from this correlation, predictions can be made of the absorption properties of probes based only on information from MD trajectories. We show experimentally that red shifts observed in the excitation maxima of LCOs when bound to amyloid protein aggregates are also evident in absorption spectra. We predict that these red shifts are due to conformational restriction of the LCO in a protein binding pocket, causing a planarization of the conjugated backbone. On the basis of our studies of planarity, it is shown that such shifts are both possible and realistic.

Pentameric thiophene-based ligands that spectrally discriminate amyloid-ß and tau aggregates display distinct solvatochromism and viscosity-induced spectral shifts.

A wide range of neurodegenerative diseases are characterized by the deposition of multiple protein aggregates. Ligands for molecular characterization and discrimination of these pathological hallmarks are thus important for understanding their potential role in pathogenesis as well as for clinical diagnosis of the disease. In this regard, luminescent conjugated oligothiophenes (LCOs) have proven useful for spectral discrimination of amyloid-beta (Aß) and tau neurofibrillary tangles (NFTs), two of the pathological hallmarks associated with Alzheimer's disease. Herein, the solvatochromism of a library of anionic pentameric thiophene-based ligands, as well as their ability to spectrally discriminate Aß and tau aggregates, were investigated. Overall, the results from this study identified distinct solvatochromic and viscosity-dependent behavior of thiophene-based ligands that can be applied as indices to direct the chemical design of improved LCOs for spectral separation of Aß and tau aggregates in brain tissue sections. The results also suggest that the observed spectral transitions of the ligands are due to their ability to conform by induced fit to specific microenvironments within the binding interface of each particular protein aggregate. We foresee that these findings might aid in the chemical design of thiophene-based ligands that are increasingly selective for distinct disease-associated protein aggregates.

Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates.

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of Aß1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimer's diease brain tissue (Aß plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.

Cross ß-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions.

BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steatohepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross ß-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross ß-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, α1-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross ß-sheet conformation than K18, whereas p62 never had cross ß-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly α-helical to cross ß-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8⁻/⁻ mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.

A fluorescent pentameric thiophene derivative detects in vitro-formed prefibrillar protein aggregates.

Protein aggregation is associated with a wide range of diseases, and molecular probes that are able to detect a diversity of misfolded protein assemblies are of great importance. The identification of prefibrillar states preceding the formation of well-defined amyloid fibrils is of particular interest both because of their likely role in the mechanism of fibril formation and because of the growing awareness that these species are likely to play a critical role in the pathogenesis of protein deposition diseases. Herein, we explore the use of an anionic oligothiophene derivative, p-FTAA, for detection of prefibrillar protein aggregates during in vitro fibrillation of three different amyloidogenic proteins (insulin, lysozyme, and prion protein). p-FTAA generally detected prefibrillar protein aggregates that could not be detected by thioflavine T fluorescence and in addition showed high fluorescence when bound to mature fibrils. Second, the kinetics of protein aggregation or the formation of amyloid fibrils of insulin was not extensively influenced by the presence of various concentrations of p-FTAA. These results establish the use of p-FTAA as an additional tool for studying the process of protein aggregation.

Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.

Amyloids are highly organized cross-beta-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function, as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. We found that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross-beta-sheet-rich conformation. Thus, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.

One-bead-one-compound library of end-capped dipeptides and deconvolution by microflow NMR.

As part of our program to identify novel small molecules with interesting biological activity, we have designed and synthesized a library of end-capped dipeptides with an emphasis on compound diversity, complexity, and membrane permeability. An approximately 1500-member library was synthesized manually on large polystyrene beads using the mix-and-split method. The final compounds were cleaved into 384-well plates to generate individual stock solutions for input into high-throughput biological screens. Individual compounds were decoded using a combination of mass spectrometry and microflow NMR spectroscopy. In principle, this approach to deconvolution obviates the need for complicated binary encoding-decoding strategies for one-bead-one-compound libraries.