Diseased Human Tissues Research Articles

Structure and Mechanism of Ergothionase from Treponema denticola.

Ergothioneine is a sulfur-containing histidine derivative that emerges from microbial biosynthesis and enters the human body through intestinal uptake and regulated distribution into specific tissues. Although the proteins involved in biosynthesis and uptake are well characterized, less is known about the degradative pathways of ergothioneine. This report describes the crystal structure of the active form of ergothionase from the oral pathogen Treponema denticola complexed with the substrate analogue desmethyl-ergothioneine sulfonic acid. This enzyme catalyzes the 1,2-elimination of trimethylamine from ergothioneine and ergothioneine sulfonic acid by using a unique mode of substrate activation combined with acid/base catalysis. This structural and mechanistic investigation revealed four essential catalytic residues, which are strictly conserved in homologous proteins from common gastrointestinal bacteria and numerous pathogenic bacteria, suggesting that bacterial activity may play an important role in determining the availability of ergothioneine in healthy and diseased human tissue.

Somatic LINE-1 retrotransposition in cortical neurons and non-brain tissues of Rett patients and healthy individuals.

Mounting evidence supports that LINE-1 (L1) retrotransposition can occur postzygotically in healthy and diseased human tissues, contributing to genomic mosaicism in the brain and other somatic tissues of an individual. However, the genomic distribution of somatic human-specific LINE-1 (L1Hs) insertions and their potential impact on carrier cells remain unclear. Here, using a PCR-based targeted bulk sequencing approach, we profiled 9,181 somatic insertions from 20 postmortem tissues from five Rett patients and their matched healthy controls. We identified and validated somatic L1Hs insertions in both cortical neurons and non-brain tissues. In Rett patients, somatic insertions were significantly depleted in exons—mainly contributed by long genes—than healthy controls, implying that cells carrying MECP2 mutations might be defenseless against a second exonic L1Hs insertion. We observed a significant increase of somatic L1Hs insertions in the brain compared with non-brain tissues from the same individual. Compared to germline insertions, somatic insertions were less sense-depleted to transcripts, indicating that they underwent weaker selective pressure on the orientation of insertion. Our observations demonstrate that somatic L1Hs insertions contribute to genomic diversity and MeCP2 dysfunction alters their genomic patterns in Rett patients.

The Pathobiology of TDP-43 C-Terminal Fragments in ALS and FTLD.

During neurodegenerative disease, the multifunctional RNA-binding protein TDP-43 undergoes a vast array of post-translational modifications, including phosphorylation, acetylation, and cleavage. Many of these alterations may directly contribute to the pathogenesis of TDP-43 proteinopathies, which include most forms of amyotrophic lateral sclerosis (ALS) and approximately half of all frontotemporal dementia, pathologically identified as frontotemporal lobar degeneration (FTLD) with TDP-43 pathology. However, the relative contributions of the various TDP-43 post-translational modifications to disease remain unclear, and indeed some may be secondary epiphenomena rather than disease-causative. It is therefore critical to determine the involvement of each modification in disease processes to allow the design of targeted treatments. In particular, TDP-43 C-terminal fragments (CTFs) accumulate in the brains of people with ALS and FTLD and are therefore described as a neuropathological signature of these diseases. Remarkably, these TDP-43 CTFs are rarely observed in the spinal cord, even in ALS which involves dramatic degeneration of spinal motor neurons. Therefore, TDP-43 CTFs are not produced non-specifically in the course of all forms of TDP-43-related neurodegeneration, but rather variably arise due to additional factors influenced by regional heterogeneity in the central nervous system. In this review, we summarize how TDP-43 CTFs are generated and degraded by cells, and critique evidence from studies of TDP-43 CTF pathology in human disease tissues, as well as cell and animal models, to analyze the pathophysiological relevance of TDP-43 CTFs to ALS and FTLD. Numerous studies now indicate that, although TDP-43 CTFs are prevalent in ALS and FTLD brains, disease-related pathology is only variably reproduced in TDP-43 CTF cell culture models. Furthermore, TDP-43 CTF expression in both transgenic and viral-mediated in vivo models largely fails to induce motor or behavioral dysfunction reminiscent of human disease. We therefore conclude that although TDP-43 CTFs are a hallmark of TDP-43-related neurodegeneration in the brain, they are not a primary cause of ALS or FTLD.

FASN-Dependent Metabolism Links Neural Stem Cell Activity to Intellectual Disability

Failure of neural stem cell (NSC) activity and subsequently neurogenesis during brain development has been linked to cognitive impairment and intellectual disability. However, it remains unclear if changes in metabolism, recently discovered as a key regulator of somatic stem cell activity, contribute to altered neurogenesis and cognitive deficits in humans. To investigate a link between NSC-associated lipid metabolism and cognition we generated mice and human embryonic stem cells (hESCs) mimicking a variant in fatty acid synthase (FASN; R1819W), a metabolic regulator of rodent NSC activity recently identified in humans with intellectual disability. Strikingly, mice homozygous for the FASN R1812W variant have impaired hippocampal NSC activity associated with cognitive impairment due to presumed toxic accumulation of lipids in NSCs and subsequent lipogenic ER stress. Moreover, human NSCs (hNSCs) homozygous for the FASN R1819W variant show reduced rates of proliferation in 2D cultures and 3D forebrain regionalized organoids, revealing that the functional significance of lipid metabolism for NSC activity is conserved between rodents and humans. By taking a comprehensive disease modeling approach, using mouse and human tissue genome engineering, our data provide the first genetic evidence for a link between altered lipid metabolism, NSC activity and brain function in humans.

Proteome Imaging: From Classic to Modern Mass Spectrometry-Based Molecular Histology.

In order to overcome the limitations of classic imaging in Histology during the actually era of multiomics, the multi-color "molecular microscope" by its emerging "molecular pictures" offers quantitative and spatial information about thousands of molecular profiles without labeling of potential targets. Healthy and diseased human tissues, as well as those of diverse invertebrate and vertebrate animal models, including genetically engineered species and cultured cells, can be easily analyzed by histology-directed MALDI imaging mass spectrometry. The aims of this review are to discuss a range of proteomic information emerging from MALDI mass spectrometry imaging comparative to classic histology, histochemistry and immunohistochemistry, with applications in biology and medicine, concerning the detection and distribution of structural proteins and biological active molecules, such as antimicrobial peptides and proteins, allergens, neurotransmitters and hormones, enzymes, growth factors, toxins and others. The molecular imaging is very well suited for discovery and validation of candidate protein biomarkers in neuroproteomics, oncoproteomics, aging and age-related diseases, parasitoproteomics, forensic, and ecotoxicology. Additionally, in situ proteome imaging may help to elucidate the physiological and pathological mechanisms involved in developmental biology, reproductive research, amyloidogenesis, tumorigenesis, wound healing, neural network regeneration, matrix mineralization, apoptosis and oxidative stress, pain tolerance, cell cycle and transformation under oncogenic stress, tumor heterogeneity, behavior and aggressiveness, drugs bioaccumulation and biotransformation, organism's reaction against environmental penetrating xenobiotics, immune signaling, assessment of integrity and functionality of tissue barriers, behavioral biology, and molecular origins of diseases. MALDI MSI is certainly a valuable tool for personalized medicine and "Eco-Evo-Devo" integrative biology in the current context of global environmental challenges.

NEAT1 and paraspeckles in neurodegenerative diseases: A missing lnc found?

Neurodegenerative diseases are among the most common causes of disability worldwide. Although neurodegenerative diseases are heterogeneous in both their clinical features and the underlying physiology, they are all characterised by progressive loss of specific neuronal populations. Recent experimental evidence suggests that long non-coding RNAs (lncRNAs) play important roles in the CNS in health and disease. Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) is an abundant, ubiquitously expressed lncRNA, which forms a scaffold for a specific RNA granule in the nucleus, or nuclear body, the paraspeckle. Paraspeckles act as molecular hubs for cellular processes commonly affected by neurodegeneration. Transcriptomic analyses of the diseased human tissue have revealed altered NEAT1 levels in the CNS in major neurodegenerative disorders as well as in some disease models. Although it is clear that changes in NEAT1 expression (and in some cases, paraspeckle assembly) accompany neuronal damage, our understanding of NEAT1 contribution to the disease pathogenesis is still rudimentary. In this review, we have summarised the available knowledge on NEAT1 involvement in the molecular processes linked to neurodegeneration and on NEAT1 dysregulation in this type of disease, with a special focus on amyotrophic lateral sclerosis. The goal of this review is to attract the attention of researchers in the field of neurodegeneration to NEAT1 and paraspeckles.

Histone Variant H2A.Z Is Required for the Maintenance of Smooth Muscle Cell Identity as Revealed by Single-Cell Transcriptomics.

Histone variants endow chromatin with specific structures, and play essential roles in development and diseases. However, little is known about their roles in controlling cell identity in vascular diseases. Given the cell heterogeneity in atherosclerotic lesions, we applied single-cell RNA-Sequencing to analyze diseased human arteries, and identified histone variant H2A.Z as a key histone signature to maintain vascular smooth muscle cell (VSMC) identity. We show that H2A.Z occupies genomic regions near VSMC marker genes, and its occupancy is decreased in VSMCs undergoing dedifferentiation. Mechanistically, H2A.Z occupancy preferentially promotes nucleosome turnover, and facilitates the recruitment of SMAD3 and MED1, thereby activating VSMC marker gene expression. In addition, H2A.Z expression is dramatically reduced at both mRNA and protein levels in diseased human vascular tissues compared to those in normal arteries. Notably, in vivo overexpression of H2A.Z rescues injury-induced loss of VSMC identity and neointima formation. Together, our data introduce dynamic occupancy of a histone variant as a novel regulatory basis contributing to cell fate decisions, and imply H2A.Z as a potential intervention node for vascular diseases.

Capturing complexity of the diffusion-weighted MR signal decay

Diffusion-weighted MRI (dMRI) is a key component of clinical radiology. When analyzing diffusion-weighted images, radiologists often seek to infer microscopic tissue structure through measurements of the diffusion coefficient, D0 (mm2/s). This multi-scale problem is framed by the creation of diffusion models of signal decay based on physical laws, histological structure, and biophysical constraints. The purpose of this paper is to simplify the model building process by focusing on the observed decay in the effective diffusion coefficient as a function of diffusion weighting (b-value), D(b), that is often observed in complex biological tissues. We call this approach the varying diffusion curvature (VDC) model. Since this is a heuristic model, the exact functional form of this decay is not important, so here we examine a simple exponential function, D(b) = D0exp(−bD1), where D0 and D1 capture aspects of hindered and restricted diffusion, respectively. As an example of the potential of the VDC model, we applied it to dMRI data collected from normal and diseased human brain tissue using Stejskal-Tanner diffusion gradient pulses. In order to illustrate the connection between D0 and D1 and the sub-voxel structure we also analyzed dMRI data from families of Sephadex beads selected with increasing tortuosity. Finally, we applied the VDC model to dMRI simulations of nested muscle fiber phantoms whose permeability, atrophy, and fiber size distribution could be changed. These results demonstrate that the VDC model is sensitive to sub-voxel tissue structure and composition (porosity, tortuosity, and permeability), hence can capture tissue complexity in a manner that could be easily applied in clinical dMRI.

Protein network analysis reveals selectively vulnerable regions and biological processes in FTD.

ObjectiveThe neuroanatomical profile of behavioral variant frontotemporal dementia (bvFTD) suggests a common biological etiology of disease despite disparate pathologic causes; we investigated the genetic underpinnings of this selective regional vulnerability to identify new risk factors for bvFTD.MethodsWe used recently developed analytical techniques designed to address the limitations of genome-wide association studies to generate a protein interaction network of 63 bvFTD risk genes. We characterized this network using gene expression data from healthy and diseased human brain tissue, evaluating regional network expression patterns across the lifespan as well as the cell types and biological processes most affected in bvFTD.ResultsWe found that bvFTD network genes show enriched expression across the human lifespan in vulnerable neuronal populations, are implicated in cell signaling, cell cycle, immune function, and development, and are differentially expressed in pathologically confirmed frontotemporal lobar degeneration cases. Five of the genes highlighted by our differential expression analyses, BAIAP2, ERBB3, POU2F2, SMARCA2, and CDC37, appear to be novel bvFTD risk loci.ConclusionsOur findings suggest that the cumulative burden of common genetic variation in an interacting protein network expressed in specific brain regions across the lifespan may influence susceptibility to bvFTD.

The long non-coding RNA NEAT1 is elevated in polyglutamine repeat expansion diseases and protects from disease gene-dependent toxicities.

Polyglutamine (polyQ) repeat diseases are a class of neurodegenerative disorders caused by CAG-repeat expansion. There are diverse cellular mechanisms behind the pathogenesis of polyQ disorders, including transcriptional dysregulation. Interestingly, we find that levels of the long isoform of nuclear paraspeckle assembly transcript 1 (Neat1L) are elevated in the brains of mouse models of spinocerebellar ataxia types 1, 2, 7 and Huntington's disease (HD). Neat1L was also elevated in differentiated striatal neurons derived from HD knock-in mice and in HD patient brains. The elevation was mutant Huntingtin (mHTT) dependent, as knockdown of mHTT in vitro and in vivo restored Neat1L to normal levels. In additional studies, we found that Neat1L is repressed by methyl CpG binding protein 2 (MeCP2) by RNA-protein interaction but not by occupancy of MeCP2 at its promoter. We also found that NEAT1L overexpression protects from mHTT-induced cytotoxicity, while reducing it enhanced mHTT-dependent toxicity. Gene set enrichment analysis of previously published RNA sequencing data from mouse embryonic fibroblasts and cells derived from HD patients shows that loss of NEAT1L impairs multiple cellular functions, including pathways involved in cell proliferation and development. Intriguingly, the genes dysregulated in HD human brain samples overlap with pathways affected by a reduction in NEAT1, confirming the correlation of NEAT1L and HD-induced perturbations. Cumulatively, the role of NEAT1L in polyQ disease model systems and human tissues suggests that it may play a protective role in CAG-repeat expansion diseases.

Abstract 236: Histone Variant H2A.Z Defines Cell Identity in Smooth Muscle Cells as Revealed by Single-Cell Transcriptomics

Background: Histone variants endow chromatin with specific structures, and play essential roles in development and diseases. However, little is known about their roles in controlling cell identity in vascular diseases. Methods and Results: Given the cell heterogeneity in atherosclerotic lesions, we applied single-cell RNA-Seq to analyze human diseased arteries, and identified histone variant H2A.Z as a key histone signature to maintain vascular smooth muscle cell (VSMC) identity. We show that H2A.Z occupies genomic regions near VSMC marker genes and its occupancy is decreased in VSMC undergoing dedifferentiation. Mechanistically, H2A.Z occupancy preferentially promotes nucleosome turnover, facilitates the recruitment of Smad3 and Med1, thereby activating VSMC marker gene expression. In human diseased vascular tissue, H2A.Z expression is dramatically decreased. Notably, in vivo overexpression of H2A.Z rescued injury-induced loss of VSMC identity and neointima formation. Conclusions: Together, our data introduce dynamic occupancy of a histone variant as a novel regulatory basis contributing to cell fate decisions, and imply H2A.Z as a potential intervention node for vascular diseases.

Abstract 1425: A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples

Abstract Next-generation sequencing (NGS) methods are used extensively to profile mutations present in diseased human tissues. These genomic approaches hold great promise for personalized medicine but sequencing accuracy is essential for proper patient diagnosis and determining a treatment plan. A common source of DNA for genomic profiling is formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from patient biopsy. FFPE DNA poses important challenges for preparing NGS libraries including low input amounts and poor DNA quality, resulting from extensive fixation- and storage-induced DNA damage. Additionally, these damage-induced sequencing artifacts raise the background level of mutations, making it difficult to discern true, low frequency, disease-causing variants from noise. We previously showed that a major fraction of somatic mutations described in publicly available datasets are due to such sequencing artifacts (Chen et al., Science 2017). Furthermore, we showed that enzymatic repair of DNA before library preparation improves the library quality and reduces background noise. We developed a second-generation DNA repair enzyme mix (V2) that efficiently repairs the most prevalent damage types found in FFPE DNA and further improves the quality and yield of NGS libraries. Additionally, we tested the efficacy of the V2 repair mix in improving sequencing accuracy for FFPE DNA samples obtained from different cancer tissues. We performed target enrichment on a panel of 151 cancer genes, deep sequenced, and performed variant analysis. For a subset of variants, we further validated our results using a droplet digital PCR (ddPCR) assay. Both methods showed that the V2 repair mix did not alter the overall frequency of variants identified, thus it did not introduce bias, but significantly improved the sequencing accuracy by reducing the number of false variant calls. Therefore, enzymatic repair is a critical first step in preparing FFPE DNA sequencing libraries, allowing more sensitive and robust detection of low frequency, disease variants. Citation Format: Pingfang Liu, Margaret Heider, Chen Song, Lixin Chen, Laurence Ettwiller, Lauren Higgins, Eileen Dimalanta, Theodore Davis, Thomas Evans. A multi-enzyme DNA repair mix improves library quality and sequencing accuracy in FFPE tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1425.

Biomaterials and heart recovery: cardiac repair, regeneration and healing in the MCS era: a state of the "heart".

Regenerative medicine is an emerging interdisciplinary field of scientific research that, supported by tissue engineering is, nowadays, a valuable and reliable solution dealing with the actual organs shortage and the unresolved limits of biological or prosthetic materials used in repair and replacement of diseased or damaged human tissues and organs. Due to the improvements in design and materials, and to the changing of clinical features of patients treated for valvular heart disease the distance between the ideal valve and the available prostheses has been shortened. We will then deal with the developing of new tools aiming at replacing or repair cardiac tissues that still represent an unmet clinical need for the surgeons and indeed for their patients. In the effort of improving treatment for the cardiovascular disease (CVD), scientists struggle with the lack of self-regenerative capacities of finally differentiated cardiovascular tissues. In this context, using several converging technological approaches, regenerative medicine moves beyond traditional transplantation and replacement therapies and can restore tissue impaired function. It may also play an essential role in surgery daily routine, leading to produce devices such as injectable hydrogels, cardiac patches, bioresorbable stents and vascular grafts made by increasingly sophisticated biomaterial scaffolds; tailored devices promptly fabricated according to surgeon necessity and patient anatomy and pathology will hopefully represent a daily activity in the next future. The employment of these devices, still far from the in vitro reproduction of functional organs, has the main aim to achieve a self-renewal process in damaged tissues simulating endogenous resident cell populations. In this field, the collaboration and cooperation between cardiothoracic surgeons and bioengineers appear necessary to modify these innovative devices employed in preclinical studies according to the surgeon's needs.

FEASIBILITY STUDY OF SYNCHROTRON-BASED MICROTOMOGRAPHY TO IDENTIFY α-SYNUCLEIN OLIGOMERS IN POSTMORTEM TISSUE

Background Lewy body disease (LBD) is the second most common cause of neurodegenerative dementia after Alzheimer’s disease and presents a challenge to healthcare services in an ageing society because current therapeutic approaches do not halt the disease’s progress. It has been suggested that aggregation of the protein α-synuclein plays a critical role in LBD. One pathway for aggregation involves the creation of low molecular weight α-synuclein oligomers, which have been observed to induce cell death. Precise location of Lewy bodies and their precursors could allow researchers to better relate their presence to clinical symptoms as well as infer their physiological effects. While conventional light microscopy of histological slices can visualize Lewy bodies and larger protein aggregates, it provides only two-dimensional information and diffraction-limited resolution prevents visualization of the smaller (approximately 50 nanometer) protein bundles. For this reason, we propose hard X-ray micro- and nano-tomography modalities for complementary, non-destructive, three-dimensional visualization of brain tissues taking advantage of developments at synchrotron radiation facilities. Methods As a proof-of-concept, we imaged paraffin-embedded fusiform gyrus tissue (provided by Newcastle Brain Tissue Resource) from a DLB case at the European Synchrotron Radiation Facility (ESRF Grenoble, France provided beamtime at ID19 from proposal MD-1055) using single-distance inline phase-contrast X-ray tomography with pixel size of 1.6 micrometers. A H&E stained histology slice of the same sample was also taken. Results A correspondence between the micro computed tomography and histology datasets could be made by tracking the orientation of vessels in the specimen. The micro computed tomography demonstrated as shown in figure (left) sufficient contrast and resolution for identification of the lower cortical layer as well as visualization of cells in three-dimensional manner, with confirmation from the annotated histology slice. Conclusions The present work serves as a proof-of-concept for the complementary use of phase contrast X-ray tomography and histology in the investigation of neurodegenerative diseases down to the cellular level. These results open the door for further nanometer resolution studies. Opens large image Nearby slices from phase-contrast microcomputed tomography (μ-CT) (left) and H&E stained histology (right) of diseased human brain tissue. The modalities can be used in complement, with the non-destructive μ-CT directing cutting planes for histological sectioning and extending histology into the third dimension.

Abstract 2187: A rapid autopsy program for cancer research

Abstract Many new technologies, such as genomics, transcriptomics, proteomics and metabolomics, are directed at molecular-level analyses of high-quality human normal and diseased tissues. At present, however, the relative lack of suitable tissue for this work is a critical roadblock to the full utilization of these new methods. Tissue is traditionally collected at the time of therapeutic surgical interventions, such as biopsy, or at the time of death, by autopsy. Biopsy is only done on individuals with disease and therefore normal control tissues are only obtained by chance. Biopsy tissue is often completely used for diagnostic purposes or is insufficient in quantity to constitute a shared resource, or for studies of intra-tumoral heterogeneity. Metastatic tissue is often not biopsied, precluding analysis of its evolving molecular changes. Both diseased and normal control tissue could potentially be obtained at autopsy, but autopsy tissue is generally only suitable when it is rapidly obtained after death. The Brain and Body Donation Program (BBDP) in Sun City, Arizona, is a not-for-profit longitudinal clinicopathological study of aging and is the world's only consistently-rapid autopsy program (3 h median). Board-certified pathologists diagnose all tissue microscopically. Many cases have both primary and metastatic tumor tissue. Both fresh-frozen & formalin-fixed samples are saved. All subjects are research volunteers with comprehensive clinical documentation. Our informed consent allows wide sharing of tissue and data, including with for-profit companies. More than 100 cancer autopsies have been done; fixed and frozen tissue is available from more than 25 cancer types, many of which have both primary and metastatic tumor tissue. Normal control tissue is available from more than 40 tissue types and RNA quality is high (Walker DG et al, Cell Tissue Bank 2016 17(3):361-75). The BBDP is listed on the NCI Specimen Resource Locator, and inquiries may be made directly to our website, www.brainandbodydonationprogram.org. Citation Format: Thomas G. Beach, Lucia I. Sue, Geidy E. Serrano, Anthony Intorcia, Jessica Walker, Michael Glass, Michael Callan. A rapid autopsy program for cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2187.

Comparison of microwave dielectric properties of human normal, benign and malignant thyroid tissues obtained from surgeries: a preliminary study

The aim of this work was to investigate whether there are differences in electromagnetic properties between normal and variously pathological human thyroid tissues. Dielectric properties of normal, diseased and malignant human thyroid tissues have been investigated at microwave frequencies from 200 MHz to 10 GHz; diseased tissues involved goiters, thyroiditis and adenomatous nodules. Measurements were carried out on freshly excised thyroid samples from 14 surgery patients within less than one hour from excision, using the open-ended coaxial probe technique. Complex permittivity values of each measured specimen were fitted to a 2-pole Cole–Cole model. Thyroid samples were classified into five categories according to their final histological examination: normal, struma (nodular goiter), thyroiditis, adenoma and cancer. Data analysis showed that the dielectric constant of a normal thyroid is on average 10% lower than relative permittivity of cancer and less than 8% lower than that of goiter over the entire frequency band. Conductivity for normal samples is from 21% to 8% lower than that for malignant lesions and from 14% to 7% lower than conductivity of goiter (struma) in the band ranging from 0.2 to 2.45 GHz. Above this frequency range no significant difference is found in terms of electrical conductivity. No significant contrast has, however, been found between dielectric properties of cancer and other benign pathologies affecting the gland. These findings are relevant for microwave diagnosis endeavours and for all biomedical applications involving the electrical properties of human tissues.

DNA methylation, through DNMT1, has an essential role in the development of gastrointestinal smooth muscle cells and disease

DNA methylation is a key epigenetic modification that can regulate gene expression. Genomic DNA hypomethylation is commonly found in many gastrointestinal (GI) diseases. Dysregulated gene expression in GI smooth muscle cells (GI-SMCs) can lead to motility disorders. However, the consequences of genomic DNA hypomethylation within GI-SMCs are still elusive. Utilizing a Cre-lox murine model, we have generated SMC-restricted DNA methyltransferase 1 (Dnmt1) knockout (KO) mice and analyzed the effects of Dnmt1 deficiency. Dnmt1-KO pups are born smaller than their wild-type littermates, have shortened GI tracts, and lose peristaltic movement due to loss of the tunica muscularis in their intestine, causing massive intestinal dilation, and death around postnatal day 21. Within smooth muscle tissue, significant CpG hypomethylation occurs across the genome at promoters, introns, and exons. Additionally, there is a marked loss of differentiated SMC markers (Srf, Myh11, miR-133, miR-143/145), an increase in pro-apoptotic markers (Nr4a1, Gadd45g), loss of cellular connectivity, and an accumulation of coated vesicles within SMC. Interestingly, we observed consistent abnormal expression patterns of enzymes involved in DNA methylation between both Dnmt1-KO mice and diseased human GI tissue. These data demonstrate that DNA hypomethylation in embryonic SMC, via congenital Dnmt1 deficiency, contributes to massive dysregulation of gene expression and is lethal to GI-SMC. These results suggest that Dnmt1 has a necessary role in the embryonic, primary development process of SMC with consistent patterns being found in human GI diseased tissue.

Metabololipidomic profiling of functional immunoresolvent clusters and eicosanoids in mammalian tissues

Metabolomics enables a systems approach to interrogate the bioactive mediators, their pathways and further metabolites involved in the physiology and pathophysiology of human and animal tissues. New metabololipidomic approaches with mass spectrometry presented in this brief review can now be utilized for the identification and profiling of lipid mediator networks that control inflammation-resolution in human blood and healthy and diseased solid tissues. Coagulation of blood is a protective response that prevents excessive bleeding on injury of blood vessels. Here, we review novel approaches to understand the relationship(s) between coagulation and resolution of inflammation and infection. To determine whether coagulation is involved in host-protective actions by lipid mediators, we used a metabololipidomic-based profiling approach with human whole blood (WB) during coagulation. We identified recently temporal clusters of endogenously produced pro-thrombotic and proinflammatory lipid mediators (eicosanoids), as well as specialized proresolving mediators (SPMs) in this vital process. In addition to the classic eicosanoids (prostaglandins, thromboxanes and leukotrienes), a specific SPM cluster was identified that consists of resolvin E1 (RvE1), RvD1, RvD5, lipoxin B4, and maresin 1, each of which present at bioactive concentrations (0.1–1 nM). The removal of adenosine from coagulating blood samples significantly enhances SPM amounts and unleashes the biosynthesis of RvD3, RvD4, and RvD6 evident following rapid snap freezing with centrifugation before extraction and LC-MS-MS. The classic cyclooxygenase inhibitors, celecoxib and indomethacin, that block thromboxanes and prostanoids do not block production of the clot-driven SPM cluster. Unbiased mass cytometry analysis demonstrated that the SPM cluster produced in human blood targets leukocytes at the single-cell level, directly activating extracellular signaling in human neutrophils and monocytes. Human whole blood treated with the components of this SPM cluster enhanced both phagocytosis and killing of Escherichia coli by leukocytes. Thus, we identified a pro-resolving lipid mediator circuit and specific SPM cluster that promotes host defense. This new lipid mediator (LM)-SPM metabololipidomic approach now provides accessible metabolomic profiles in healthy and diseased human tissues, including cancer, for precision and personalized medicine.

Polydimethylsiloxane Composites for Optical Ultrasound Generation and Multimodality Imaging

AbstractPolydimethylsiloxane (PDMS) is widely used in biomedical science and can form composites that have broad applicability. One promising application where PDMS composites offer several advantages is optical ultrasound generation via the photoacoustic effect. Here, methods to create these PDMS composites are reviewed and classified. It is highlighted how the composites can be applied to a range of substrates, from micrometer‐scale, temperature‐sensitive optical fibers to centimeter‐scale curved and planar surfaces. The resulting composites have enabled all‐optical ultrasound imaging of biological tissues both ex vivo and in vivo, with high spatial resolution and with clinically relevant contrast. In addition, the first 3D all‐optical pulse‐echo ultrasound imaging of ex vivo human tissue, using a PDMS‐multiwalled carbon nanotube composite and a fiber‐optic ultrasound receiver, is presented. Gold nanoparticle‐PDMS and crystal violet‐PDMS composites with prominent absorption at one wavelength range for pulse‐echo ultrasound imaging and transmission at a second wavelength range for photoacoustic imaging are also presented. Using these devices, images of diseased human vascular tissue with both structural and molecular contrast are obtained. With a broader perspective, literature on recent advances in PDMS microfabrication from different fields is highlighted, and methods for incorporating them into new generations of optical ultrasound generators are suggested.

Markers of oxidative damage to lipids, nucleic acids and proteins and antioxidant enzymes activities in Alzheimer's disease brain: A meta-analysis in human pathological specimens.

Oxidative stress and decreased cellular responsiveness to oxidative stress are thought to influence brain aging and Alzheimer's disease, but the specific patterns of oxidative damage and the underlying mechanism leading to this damage are not definitively known. The objective of this study was to define the pattern of changes in oxidative-stress related markers by brain region in human Alzheimer's disease and mild cognitive impairment brain tissue. Observational case-control studies were identified from systematic queries of PubMed, ISI Web of Science and Scopus databases and studies were evaluated with appropriate quality measures. The data was used to construct a region-by-region meta-analysis of malondialdehyde, 4-hydroxynonenal, protein carbonylation, 8-hydroxyguanine levels and superoxide dismutase, glutathione peroxidase, glutathione reductase and catalase activities. We also evaluated ascorbic acid, tocopherol, uric acid and glutathione levels. The analysis was complicated in several cases by publication bias and/or outlier data. We found that malondialdehyde levels were slightly increased in the temporal and occipital lobes and hippocampus, but this analysis was significantly impacted by publication bias. 4-hydroxynonenal levels were unchanged in every brain region. There was no change in 8-hydroxyguanine level in any brain region and protein carbonylation levels were unchanged except for a slight increase in the occipital lobe. Superoxide dismutase, glutathione peroxidase and reductase and catalase activities were not decreased in any brain region. There was limited data reporting non-enzymatic antioxidant levels in Alzheimer's disease brain, although glutathione and tocopherol levels appear to be unchanged. Minimal quantitative data is available from brain tissue from patients with mild cognitive impairment. While there is modest evidence supporting minor regional changes in markers of oxidative damage, this analysis fails to identify a consistent pattern of pro-oxidative changes and accumulation of oxidative damage in bulk tissue analysis in the setting of Alzheimer's disease, as has been widely reported.