Hernadez-Perez OR, Hernandez VS, Nava-Kopp A, Barrio RA, Seifi M, Swinny JD, Eiden LE, Zhang L.
PMID: - | DOI: 10.3389/fnins.2019.00196
The locus coeruleus (LC)-norepinephrine (NE) system modulates a range of salient brain functions, including memory and response to stress. The LC-NE system is regulated by neurochemically diverse inputs, including a range of neuropeptides such as arginine-vasopressin (AVP). Whilst the origins of many of these LC inputs, their synaptic connectivity with LC neurons, and their contribution to LC-mediated brain functions, have been well characterized, this is not the case for the AVP-LC system. Therefore, our aims were to define the types of synapses formed by AVP+ fibers with LC neurons using immunohistochemistry together with confocal and transmission electron microscopy (TEM), the origins of such inputs, using retrograde tracers, and the plasticity of the LC AVP system in response to stress and spatial learning, using the maternal separation (MS) and Morris water maze (MWM) paradigms respectively, in rat. Confocal microscopy revealed that AVP+ fibers contacting tyrosine hydroxylase (TH)+ LC neurons were also immunopositive for vesicular glutamate transporter 2, a marker of presynaptic glutamatergic axons. TEM confirmed that AVP+ axons formed Gray type I (asymmetric) synapses with TH+ dendrites thus confirming excitatory synaptic connections between these systems. Retrograde tracing revealed that these LC AVP+ fibers originate from hypothalamic vasopressinergic magnocellular neurosecretory neurons (AVPMN). MS induced a significant increase in the density of LC AVP+ fibers. Finally, AVPMNN circuit upregulation by water-deprivation improved MWM performance while increased Fos expression was found in LC and efferent regions such as hippocampus and prefrontal cortex, suggesting that AVPMMN projections to LC could integrate homeostatic responses modifying neuroplasticity.
Journal of immunological methods
Schafer, C;Young, D;Singh, H;Jayakrishnan, R;Banerjee, S;Song, Y;Dobi, A;Petrovics, G;Srivastava, S;Srivastava, S;Sesterhenn, IA;Chesnut, GT;Tan, SH;
PMID: 37196930 | DOI: 10.1016/j.jim.2023.113493
Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function.An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens.Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (-) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells.The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors.
Meeker, T;Tulloch, I;Kim, H;Keaser, M;Seminowicz, D;Dorsey, S;
| DOI: 10.1016/j.jpain.2023.02.067
Previous studies have demonstrated effects of racialized minority status on thermal pain sensitivity, sensibility, and tolerance. However, there is limited evidence demonstrating effects of minority status on painful punctate mechanical stimuli and self-report pain. We analyzed the effects of racialized minority status on heat pain sensitivity, sensibility to painful heat and punctate mechanical stimuli, and Pain Sensitivity Questionnaire (PSQ) scores. Our secondary purpose was to test face validity of the PSQ in a US population. Using quantitative sensory testing for painful heat and punctate mechanical stimuli (forces: 64, 128, 256 and 512 mN), and self-report PSQ, we evaluated pain sensitivity and sensibility in 134 healthy participants (34 Asian, 25 Black, and 75 White). We used linear mixed models to analyze outcomes allowing maximal inclusion of incomplete data sets. Racialized minority status was associated with greater heat pain sensitivity (F=7.63; p=0.00074) and PSQ scores (F=15.45; p=9.84 × 10-7) but had no effect on painful suprathreshold heat (model improvement by addition of race: Χ2=2.199; p=0.333) or punctate mechanical stimuli (F=1.50; p=0.229). Face validity of the PSQ in racialized minorities was limited by differential experience of individual items (F=19.87; p=3.28 × 10-8). Ratings of painful suprathreshold heat (R=0.204; p=0.00020) and punctate mechanical stimuli (R=0.333; p=0.00062) positively correlated with PSQ scores. Consistent with previous research, sensitivity to painful heat was affected by racialized minority status. In contrast, there was no significant effect of racialized minority status on suprathreshold painful heat or punctate mechanical stimuli. Certain items of the PSQ are inapplicable to healthy participants from racialized minority groups. NIH National Institute of Nursing Research P30NR014129.
Mucalo, L;Jia, S;Roethle, M;Singh, A;Brousseau, D;Panepinto, J;Hessner, M;Brandow, A;
| DOI: 10.1016/j.jpain.2023.02.062
Sudden, unpredictable, severe acute pain episodes are the most common sickle cell disease (SCD) complication. Some SCD patients experience frequent pain episodes while others experience rare episodes. Knowledge of the biology driving this variability is limited. Using gene transcription analyses, we previously showed an elevated inflammatory response is associated with increased SCD pain episode frequency. We sought to replicate these findings in a larger SCD cohort and identify hub genes closely associated with increased pain frequency. We conducted plasma-induced transcription analyses in 132 SCD patients (baseline health) and 60 Black controls (4-21 years, both groups). 3028 differentially expressed genes between SCD patients and controls were retained for subsequent analyses with Weighted Gene Co-Expression Network Analysis (WGCNA). WGCNA was used to define modules (functionally grouped genes) and we correlated these modules with number of pain episodes requiring health care utilization in prior three years. Of 11 identified modules, four showed significant correlation with number of pain episodes. Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used for ontological analysis of the four significant modules and key biological processes identified were inflammatory response and cellular response to lipopolysaccharide. Cytoscape was used to construct a protein-protein interaction network and the 10 top hub genes identified in hierarchical order were: TNF, CCR5, CCR1, CCL2, CXCL2, ITGAM, CCL7, CXCL3, TLR2 and MMP9. These genes, as part of the inflammatory response, support the immune system contributes to increased pain episode frequency. Identified hub genes may be leveraged as therapeutic targets for reducing SCD pain episodes. 1R61NS114954-01.
Journal of Virus Eradication
Collins, D;Hitschfel, J;Walker, B;
| DOI: 10.1016/j.jve.2022.100202
Background: HIV infection persists predominantly within follicular helper CD4+ T cell-rich B cell follicles of lymphoid tissues. Cytotoxic CD8+ T cells, which are associated with natural control of HIV infection in peripheral blood, are relatively excluded from this niche, representing a potential barrier to cellular immunity and HIV cure. To better understand the mechanisms of HIV control within lymph nodes (LN), we investigated functionality, clonotypic compartmentalization, spatial localization, phenotypic characteristics and transcriptional profiles of LN-resident virus-specific and CXCR5-expressing follicular CD8+ T cells (fCD8) in persons who control HIV without medications. Methods: We obtained paired excisional inguinal LN biopsies and peripheral blood (PB) from 19 spontaneous HIV controllers and 17 HIV+ individuals on long-term ART. HIV-specific CD8+ T cell responses were identified by IFN-γ ELISpot and functional response to antigenic stimulation was measured by flow cytometry and CFSE-based proliferation assay. Clonotypic compartmentalization and transcriptional signatures associated with localization of HIV-specific CD8+ T cells were assessed via TCR and RNA-sequencing. Spatial relationships between ongoing viral replication and fCD8 cytotoxic effector potential in GCs were measured by HIV gagpol RNAscope and immunofluorescence on fixed LN sections. Results: Antigen-induced HIV-specific CD8+ T cell proliferation and cytolytic effector upregulation consistently distinguished spontaneous controllers from noncontrollers in PB (p=0.03) and LN (p=0.04). HIV-specific CD8+ T cells from both compartments shared TCR clonotypic composition (Morisita-Horn Similarity Index 0.8-1.0), consistent with ongoing infiltration from circulation. Migration into LNs was associated with gene signatures of inflammatory chemotaxis and antigen-induced effector function. The cytolytic effectors perforin and granzyme B were elevated among virus-specific CXCR5 + fCD8 s (p
Steinhart, M;Serdy, S;van der Valk, W;Zhang, J;Kim, J;Lee, J;Koehler, K;
| DOI: 10.2139/ssrn.3974124
Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of _in vitro_ human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of _in vitro_ development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathway-related gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue.
Direct healthcare costs of lip, oral cavity and oropharyngeal cancer in Brazil
Milani, V;Zara, ALSA;da Silva, EN;Cardoso, LB;Curado, MP;Ribeiro-Rotta, RF;
PMID: 33596233 | DOI: 10.1371/journal.pone.0246475
The efficiency of public policies includes the measurement of the health resources used and their associated costs. There is a lack of studies evaluating the economic impact of oral cancer (OC). This study aims to estimate the healthcare costs of OC in Brazil from 2008 to 2016. This is a partial economic evaluation using the gross costing top-down method, considering the direct healthcare costs related to outpatients, inpatients, intensive care units, and the number of procedures, from the perspective of the public health sector. The data were extracted from the Outpatient and Inpatient Information System of the National Health System, by diagnosis according to the 10th Revision of the International Classification of Diseases, according to sites of interest: C00 to C06, C09 and C10. The values were adjusted for annual accumulated inflation and expressed in 2018 I$ (1 I$ = R$2,044). Expenditure on OC healthcare in Brazil was I$495.6 million, which was composed of 50.8% (I$251.6 million) outpatient and 49.2% (I$244.0 million) inpatient healthcare. About 177,317 admissions and 6,224,236 outpatient procedures were registered. Chemotherapy and radiotherapy comprised the largest number of procedures (88.8%) and costs (94.9%). Most of the costs were spent on people over 50 years old (72.9%) and on males (75.6%). Direct healthcare costs in Brazil for OC are substantial. Outpatient procedures were responsible for the highest total cost; however, inpatient procedures had a higher cost per procedure. Men over 50 years old consumed most of the cost and procedures for OC. The oropharynx and tongue were the sites with the highest expenditure. Further studies are needed to investigate the cost per individual, as well as direct non-medical and indirect costs of OC.
Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo
Allweiss, L;Giersch, K;Pirosu, A;Volz, T;Muench, RC;Beran, RK;Urban, S;Javanbakht, H;Fletcher, SP;Lütgehetmann, M;Dandri, M;
PMID: 33509930 | DOI: 10.1136/gutjnl-2020-322571
Silencing of the therapeutic strategies and reducing the HBV reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo. HBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events. Both siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6. These results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.
Single-cell RNA sequencing reveals SARS-CoV-2 infection dynamics in lungs of African green monkeys
Science translational medicine
Speranza, E;Williamson, BN;Feldmann, F;Sturdevant, GL;Pérez-Pérez, L;Meade-White, K;Smith, BJ;Lovaglio, J;Martens, C;Munster, VJ;Okumura, A;Shaia, C;Feldmann, H;Best, SM;de Wit, E;
PMID: 33431511 | DOI: 10.1126/scitranslmed.abe8146
Detailed knowledge about the dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is important for uncovering the viral and host factors that contribute to coronavirus disease 2019 (COVID-19) pathogenesis. Old-World nonhuman primates recapitulate mild to moderate cases of COVID-19, thereby serving as important pathogenesis models. We compared African green monkeys inoculated with infectious SARS-CoV-2 or irradiated, inactivated virus to study the dynamics of virus replication throughout the respiratory tract. Genomic RNA from the animals inoculated with the irradiated virus was found to be highly stable, whereas subgenomic RNA, an indicator of viral replication, was found to degrade quickly. We combined this information with single-cell RNA sequencing of cells isolated from the lung and lung-draining mediastinal lymph nodes and developed new analysis methods for unbiased targeting of important cells in the host response to SARS-CoV-2 infection. Through detection of reads to the viral genome, we were able to determine that replication of the virus in the lungs appeared to occur mainly in pneumocytes, whereas macrophages drove the inflammatory response. Monocyte-derived macrophages recruited to the lungs, rather than tissue-resident alveolar macrophages, were most likely to be responsible for phagocytosis of infected cells and cellular debris early in infection, with their roles switching during clearance of infection. Together, our dataset provides a detailed view of the dynamics of virus replication and host responses over the course of mild COVID-19 and serves as a valuable resource to identify therapeutic targets.
Mancarci BO, Toker L, Tripathy SJ, Li B, Rocco B, Sibille E, Pavlidis P.
PMID: - | DOI: 10.1523/ENEURO.0212-17.2017
Establishing the molecular diversity of cell types is crucial for the study of the nervous system. We compiled a cross-laboratory database of mouse brain cell type-specific transcriptomes from 36 major cell types from across the mammalian brain using rigorously curated published data from pooled cell type microarray and single cell RNA-sequencing studies. We used these data to identify cell type-specific marker genes, discovering a substantial number of novel markers, many of which we validated using computational and experimental approaches. We further demonstrate that summarized expression of marker gene sets in bulk tissue data can be used to estimate the relative cell type abundance across samples. To facilitate use of this expanding resource, we provide a user-friendly web interface at Neuroexpresso.org.
Significance Statement Cell type markers are powerful tools in the study of the nervous system that help reveal properties of cell types and acquire additional information from large scale expression experiments. Despite their usefulness in the field, known marker genes for brain cell types are few in number. We present NeuroExpresso, a database of brain cell type specific gene expression profiles, and demonstrate the use of marker genes for acquiring cell type specific information from whole tissue expression. The database will prove itself as a useful resource for researchers aiming to reveal novel properties of the cell types and aid both laboratory and computational scientists to unravel the cell type specific components of brain disorders.
Zylka, M;McCoy, E;Park, S;Patel, R;Ryan, D;Mullen, Z;Nesbitt, J;Lopez, J;Taylor-Blake, B;Krantz, J;Hu, W;Garris, R;Lima, L;Sotocinal, S;Austin, J;Kashlan, A;Jimenez, J;Shah, S;Trocinski, A;Vanden, K;Major, R;Bazick, H;Klein, M;Mogil, J;Wu, G;
| DOI: 10.1016/j.jpain.2023.02.113
Facial grimaces are now commonly used to quantify spontaneous pain in mice and other mammalian species, but scoring remains subjective and relies on humans with very different levels of proficiency. Here, we developed a Mouse Grimace Scale (MGS) for black-coated (C57BL/6) mice consisting of four facial action units (orbitals, nose, ears, whiskers). We used this scale to generate ground truth data from over 70,000 images of black mice in different settings. With this large data set, we developed a deep neural network and cloud-based software platform called PainFace (http://painface.net) that accurately scores facial grimaces of black mice on a 0-8 scale. PainFace generates over two orders of magnitude more MGS data than humans can realistically achieve, and at superhuman speed. By analyzing the frequency distribution of grimace scores, we found that mice spent >7x more time in a high grimace state following laparotomy surgery relative to sham surgery controls. The analgesic carprofen reduced the amount of time animals spent in this high grimace state after surgery. Specific facial action unit score combinations were overrepresented following laparotomy surgery, suggesting that characteristic facial expressions are associated with a high grimace state. While this study is focused on mice, PainFace was designed to simplify, standardize, and scale up grimace analyses with many other mammalian species, including humans. This work was supported by a grant from the NINDS (R01NS114259) to M.J.Z. NSF GRFP awarded to R.P.P.
Kaptan, M;Law, C;Weber, K;Pfyffer, D;Zhang, X;Maronesy, T;Glover, G;Mackey, S;
| DOI: 10.1016/j.jpain.2023.02.065
Investigation of spontaneous- so-called‘resting-state'-activity of the central nervous system with functional magnetic resonance imaging (fMRI) holds great clinical potential to identify possible prognostic and diagnostic biomarkers for pain disorders and provides novel insights into the functional architecture of the central nervous system. Although previous resting-state studies in humans characterized functional networks of the brain and recently of the spinal cord, the resting-state networks of the entire central nervous system-delineating the interaction between the cord and the brain-have not been well characterized, possibly due to technical difficulties of corticospinal fMRI. Given the important role of ascending and descending pathways to understand disorders chronic pain disorders, here we characterize the resting-state functional connectivity networks along the whole neuroaxis in 29 healthy humans as a step prior to clinical studies. 31 brain slices and 12 cervical spinal cord slices from were acquired with a tailored fMRI sequence on a 3T system. Time courses of dorsal and ventral horns were used to map spinal cord's connection to the brain via a seed-based approach. Functional connectivity maps revealed that dorsal and ventral horn are significantly correlated with sensory and motor areas in the brain such as primary and somatosensory and motor cortices as well as with the thalamus. At the same time, we have observed that they somewhat distinct functional connectivity profiles in line with their functional segregation; frontal, occipital and insular cortices were more synchronized with ventral horn whereas caudate and thalamus appeared to be more synchronized with dorsal horn reflecting their functional division. NIH NINDS R01 NS109450.
