Wildi, N;Seuberlich, T;
PMID: 37376701 | DOI: 10.3390/v15061402
Astroviruses are small nonenveloped single-stranded RNA viruses with a positive sense genome. They are known to cause gastrointestinal disease in a broad spectrum of species. Although astroviruses are distributed worldwide, a gap in knowledge of their biology and disease pathogenesis persists. Many positive-sense single-stranded RNA viruses show conserved and functionally important structures in their 5' and 3' untranslated regions (UTRs). However, not much is known about the role of the 5' and 3' UTRs in the viral replication of HAstV-1. We analyzed the UTRs of HAstV-1 for secondary RNA structures and mutated them, resulting in partial or total UTR deletion. We used a reverse genetic system to study the production of infectious viral particles and to quantify protein expression in the 5' and 3' UTR mutants, and we established an HAstV-1 replicon system containing two reporter cassettes in open reading frames 1a and 2, respectively. Our data show that 3' UTR deletions almost completely abolished viral protein expression and that 5' UTR deletions led to a reduction in infectious virus particles in infection experiments. This indicates that the presence of the UTRs is essential for the life cycle of HAstV-1 and opens avenues for further research.
Pathogens (Basel, Switzerland)
Caspe, SG;Ewing, DA;Livingstone, M;Underwood, C;Milne, E;Sargison, ND;Wattegedera, SR;Longbottom, D;
PMID: 37375536 | DOI: 10.3390/pathogens12060846
The enzootic abortion of ewes, caused by the bacterium Chlamydia abortus (C. abortus), is one of the main causes of abortion in sheep. There are multiple contributory factors, including chlamydial growth, host immune response, and hormonal balance, that result in different pregnancy outcomes, such as abortion, the birth of weak lambs that may die, or healthy lambs. This study aimed to determine the relationship between phenotypical patterns of immune cell infiltration and different pregnancy outcomes in twin-bearing sheep (both lambs born dead; one alive and one dead; both alive) when experimentally infected with C. abortus. Both the sheep uteri and placentae were collected after parturition. All samples were analysed for specific immune cell features, including cell surface antigens and the T-regulatory (Treg) cell-associated transcription factor and cytokines, by immunohistochemistry and in situ hybridisation. Some of these immunological antigens were evaluated in ovine reproductive tissues for the first time. Differential patterns of T helper/Treg cells revealed significant group effects in the placentae. It suggests the potential role that the balance of lymphocyte subsets may play in affecting different pregnancy outcomes in C. abortus-infected sheep. The present study provides novel detailed information about the immune responses observed at the maternofoetal interface in sheep at the time of pre-term abortion or lambing.
Lan, L;Evan, T;Li, H;Hussain, A;Ruiz, EJ;Zaw Thin, M;Ferreira, RMM;Ps, H;Riising, EM;Zen, Y;Almagro, J;Ng, KW;Soro-Barrio, P;Nelson, J;Koifman, G;Carvalho, J;Nye, EL;He, Y;Zhang, C;Sadanandam, A;Behrens, A;
PMID: 35768509 | DOI: 10.1038/s41586-022-04888-7
Pancreatic ductal adenocarcinoma (PDAC) shows pronounced epithelial and mesenchymal cancer cell populations1-4. Cellular heterogeneity in PDAC is an important feature in disease subtype specification3-5, but how distinct PDAC subpopulations interact, and the molecular mechanisms that underlie PDAC cell fate decisions, are incompletely understood. Here we identify the BMP inhibitor GREM16,7 as a key regulator of cellular heterogeneity in pancreatic cancer in human and mouse. Grem1 inactivation in established PDAC in mice resulted in a direct conversion of epithelial into mesenchymal PDAC cells within days, suggesting that persistent GREM1 activity is required to maintain the epithelial PDAC subpopulations. By contrast, Grem1 overexpression caused an almost complete 'epithelialization' of highly mesenchymal PDAC, indicating that high GREM1 activity is sufficient to revert the mesenchymal fate of PDAC cells. Mechanistically, Grem1 was highly expressed in mesenchymal PDAC cells and inhibited the expression of the epithelial-mesenchymal transition transcription factors Snai1 (also known as Snail) and Snai2 (also known as Slug) in the epithelial cell compartment, therefore restricting epithelial-mesenchymal plasticity. Thus, constant suppression of BMP activity is essential to maintain epithelial PDAC cells, indicating that the maintenance of the cellular heterogeneity of pancreatic cancer requires continuous paracrine signalling elicited by a single soluble factor.
Gerstmann, K;Jurčić, N;Blasco, E;Kunz, S;de Almeida Sassi, F;Wanaverbecq, N;Zampieri, N;
PMID: 35512696 | DOI: 10.1016/j.cub.2022.04.019
From swimming to walking and flying, animals have evolved specific locomotor strategies to thrive in different habitats. All types of locomotion depend on the integration of motor commands and sensory information to generate precisely coordinated movements. Cerebrospinal-fluid-contacting neurons (CSF-cN) constitute a vertebrate sensory system that monitors CSF composition and flow. In fish, CSF-cN modulate swimming activity in response to changes in pH and bending of the spinal cord; however, their role in mammals remains unknown. We used mouse genetics to study their function in quadrupedal locomotion. We found that CSF-cN are directly integrated into spinal motor circuits. The perturbation of CSF-cN function does not affect general motor activity nor the generation of locomotor rhythm and pattern but results in specific defects in skilled movements. These results identify a role for mouse CSF-cN in adaptive motor control and indicate that this sensory system evolved a novel function to accommodate the biomechanical requirements of limb-based locomotion.
McMahan, K;Giffin, V;Tostanoski, LH;Chung, B;Siamatu, M;Suthar, MS;Halfmann, P;Kawaoka, Y;Piedra-Mora, C;Jain, N;Ducat, S;Kar, S;Andersen, H;Lewis, MG;Martinot, AJ;Barouch, DH;
PMID: 35313451 | DOI: 10.1016/j.medj.2022.03.004
The SARS-CoV-2 Omicron (B.1.1.529) variant has proven highly transmissible and has outcompeted the Delta variant in many regions of the world. Early reports have also suggested that Omicron may result in less severe clinical disease in humans. Here we show that Omicron is less pathogenic than prior SARS-CoV-2 variants in Syrian golden hamsters.Hamsters were inoculated with either SARS-CoV-2 Omicron or other SARS-CoV-2 variants. Animals were followed for weight loss, and upper and lower respiratory tract tissues were assessed for viral loads and histopathology.Infection of hamsters with the SARS-CoV-2 WA1/2020, Alpha, Beta, or Delta strains led to 4-10% weight loss by day 4 and 10-17% weight loss by day 6. In contrast, infection of hamsters with two different Omicron challenge stocks did not result in any detectable weight loss, even at high challenge doses. Omicron infection led to substantial viral replication in both the upper and lower respiratory tracts but demonstrated lower viral loads in lung parenchyma and reduced pulmonary pathology compared with WA1/2020 infection.These data suggest that the SARS-CoV-2 Omicron variant may result in robust upper respiratory tract infection but less severe lower respiratory tract clinical disease compared with prior SARS-CoV-2 variants.Funding for this study was provided by NIH grant CA260476, the Massachusetts Consortium for Pathogen Readiness, the Ragon Institute, and the Musk Foundation.
Brain : a journal of neurology
Middleton, SJ;Perini, I;Andreas C, T;Weir, GA;McCann, K;Barry, AM;Marshall, A;Lee, M;Mayo, LM;Bohic, M;Baskozos, G;Morrison, I;Löken, LS;McIntyre, S;Nagi, SS;Staud, R;Sehlstedt, I;Johnson, RD;Wessberg, J;Wood, JN;Woods, CG;Moqrich, A;Olausson, H;Bennett, DL;
PMID: 34957475 | DOI: 10.1093/brain/awab482
Patients with bi-allelic loss of function mutations in the voltage-gated sodium channel Nav1.7 present with congenital insensitivity to pain (CIP), whilst low threshold mechanosensation is reportedly normal. Using psychophysics (n = 6 CIP participants and n = 86 healthy controls) and facial EMG (n = 3 CIP participants and n = 8 healthy controls) we have found that these patients also have abnormalities in the encoding of affective touch which is mediated by the specialised afferents; C-low threshold mechanoreceptors (C-LTMRs). In the mouse we found that C-LTMRs express high levels of Nav1.7. Genetic loss or selective pharmacological inhibition of Nav1.7 in C-LTMRs resulted in a significant reduction in the total sodium current density, an increased mechanical threshold and reduced sensitivity to non-noxious cooling. The behavioural consequence of loss of Nav1.7 in C-LTMRs in mice was an elevation in the von Frey mechanical threshold and less sensitivity to cooling on a thermal gradient. Nav1.7 is therefore not only essential for normal pain perception but also for normal C-LTMR function, cool sensitivity and affective touch.
Kim, D;Jung, H;Shirai, Y;Kim, H;Kim, J;Lim, D;Mori, T;Lee, H;Park, D;Kim, H;Guo, Q;Pang, B;Qiu, W;Cao, X;Kouyama-Suzuki, E;Uemura, T;Kasem, E;Fu, Y;Kim, S;Tokunaga, A;Yoshizawa, T;Suzuki, T;Sakagami, H;Lee, K;Ko, J;Tabuchi, K;Um, J;
| DOI: 10.1016/j.biopsych.2021.12.016
Background IQSEC3, a gephyrin-binding GABAergic synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood. Methods We generated mice with conditional knockout (cKO) of Iqsec3 to examine whether altered synaptic inhibition influences hippocampus-dependent fear memory formation. In addition, electrophysiological recordings, immunohistochemistry, and behavioral assays were employed to address our question. Results We found that Iqsec3-cKO induces a specific reduction in GABAergic synapse density, GABAergic synaptic transmission, and maintenance of long-term potentiation (LTP) in the hippocampal CA1 region. In addition, Iqsec3-cKO mice exhibited impaired fear memory formation. Strikingly, Iqsec3-cKO caused abnormally enhanced activation of ribosomal P70-S6 kinase1 (S6K1)-mediated signaling in the hippocampus, but not in the cortex. Furthermore, inhibiting upregulated S6K1 signaling by expressing dominant-negative S6K1 in the hippocampal CA1 of Iqsec3-cKO mice completely rescued impaired fear learning and inhibitory synapse density, but not deficits in LTP maintenance. Lastly, upregulated S6K1 signaling was rescued by IQSEC3 wild-type, but not by an ARF-GEF inactive IQSEC3 mutant. Conclusions Our results suggest that IQSEC3-mediated balanced synaptic inhibition in the hippocampal CA1 is critical for proper formation of hippocampus-dependent fear memory.
Deletion of TNFAIP6 gene in human keratinocytes demonstrates a role for TSG-6 to retain hyaluronan inside epidermis
Evrard, C;Faway, E;De Vuyst, E;Svensek, O;De Glas, V;Bergerat, D;Salmon, M;De Backer, O;Flamion, B;Le-Buanec, H;Lambert de Rouvroit, C;Poumay, Y;
| DOI: 10.1016/j.xjidi.2021.100054
TNFα-stimulated gene 6 (TSG-6) is a soluble protein secreted in the extracellular matrix (ECM) by various cell types in response to inflammatory stimuli. TSG-6 interacts with ECM molecules, particularly hyaluronan (HA), and promotes cutaneous wound closure in mouse. Between epidermal cells, the discrete ECM contains HA and tiny amount of TSG-6. However, challenges imposed to keratinocytes in reconstructed human epidermis (RHE) revealed strong induction of TSG-6 expression, after exposure to Th2 cytokines to recapitulate the atopic dermatitis phenotype, or after fungal infection that causes secretion of cytokines and antimicrobial peptides. Following both types of challenge, enhanced release of TSG-6 happens simultaneously with increased HA production. TSG-6 deficiency in N/TERT keratinocytes was created by inactivating TNFAIP6 using CRISPR/Cas9. Some TSG-6-/- keratinocytes analyzed through scratch assays tend to migrate more slowly, but produce RHE that exhibit normal morphology and differentiation. Few significant alterations were noticed by transcriptomic analysis. Nevertheless, reduced HA content in TSG-6-/- RHE was observed, along with enhanced HA release into culture medium, and this phenotype was even more pronounced following challenging conditions. Reintroduction of cells producing TSG-6 in RHE reduced HA leakage. Our results demonstrate a role for TSG-6 in sequestering HA between epidermal cells in response to inflammation.
Spatio-temporal expression pattern and role of the tight junction protein MarvelD3 in pancreas development and function
Heymans, C;Delcorte, O;Spourquet, C;Villacorte-Tabelin, M;Dupasquier, S;Achouri, Y;Mahibullah, S;Lemoine, P;Balda, MS;Matter, K;Pierreux, CE;
PMID: 34267243 | DOI: 10.1038/s41598-021-93654-2
Tight junction complexes are involved in the establishment and maintenance of cell polarity and the regulation of signalling pathways, controlling biological processes such as cell differentiation and cell proliferation. MarvelD3 is a tight junction protein expressed in adult epithelial and endothelial cells. In Xenopus laevis, MarvelD3 morphants present differentiation defects of several ectodermal derivatives. In vitro experiments further revealed that MarvelD3 couples tight junctions to the MEKK1-JNK pathway to regulate cell behaviour and survival. In this work, we found that MarvelD3 is expressed from early developmental stages in the exocrine and endocrine compartments of the pancreas, as well as in endothelial cells of this organ. We thoroughly characterized MarvelD3 expression pattern in developing pancreas and evaluated its function by genetic ablation. Surprisingly, inactivation of MarvelD3 in mice did not alter development and differentiation of the pancreatic tissue. Moreover, tight junction formation and organization, cell polarization, and activity of the JNK-pathway were not impacted by the deletion of MarvelD3.
Visualization of Respiratory Commensal Bacteria in Context of Their Natural Host Environment
Frontiers in microbiology
Bonifacio, JPP;Schmolke, M;
PMID: 34149669 | DOI: 10.3389/fmicb.2021.678389
Commensal microbes are an integral component of mammalian physiology. 16S rRNA gene-specific next generation sequencing from DNA of total organs, swabs or lavages has revolutionized the characterization of bacterial communities in virtually every ecological niche of the body. Culturomics, next allowed the isolation and characterization of commensal bacteria in the lab and the establishment of artificial communities of bacteria, which were eventually reintroduced in model organisms. Spatial organization of microbiota within a given host environment is critical to the physiological or pathological phenotypes provoked by commensal microbiota. In situ hybridization (ISH) is a complementary technique to sequencing and culturing to visualize the presence of individual bacterial operational taxonomic unit (OTUs) in context of the colonized organ. We recently applied highly sensitive in situ RNA hybridization to detection of commensal bacteria in low abundance respiratory tract samples of mice housed under specific pathogen free conditions. This technique allows species-specific detection of living bacteria using RNAScopeTM technology, while preserving the natural environment of the organ. We here provide a detailed step-by-step protocol describing the detection of commensal lung bacteria in respiratory tissue.
Molecular Therapy: Nucleic Acid
Schrom E, Huber M, Aneja M, Dohmen C, Emrich D, Geiger J, Hasenpusch G, Herrmann-Janson A, Kretzschmann V, Mykhailyk O, Pasewald T, Oak P, Hilgendorff A, Wohlleber D, Hoymann HG, Schaudien D, Plank C, Rudolph C , Kubisch-Dohmen R.
PMID: - | DOI: 10.1016/j.omtn.2017.04.006
Changes in lifestyle and environmental conditions give rise to increasing prevalence of liver and lung fibrosis, both having poor prognosis. Promising results of recombinant ACE2 protein administration in experimental liver and lung fibrosis have been reported. However, the full potential of ACE2 may be achieved by localized translation of a membrane anchored form. For this purpose, we advanced latest RNA technology for liver and lung targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles led to liver targeted while formulation in lipid nanoparticles led to lung targeted translation of significant amounts of ACE2 protein. In summary, we provided evidence that RNA transcript therapy (RTT) is a promising approach for ACE2 based treatment of liver and lung fibrosis to be tested in fibrotic disease models.
Talukdar S, Owen BM, Song P, Hernandez G, Zhang Y, Zhou Y, Scott WT, Paratala B, Turner T, Smith A, Bernardo B, Müller CP, Tang H, Mangelsdorf DJ, Goodwin B, Kliewer SA.
PMID: 26724861 | DOI: 10.1016/j.cmet.2015.12.008.
Fibroblast growth factor 21 (FGF21) is a hormone induced by various metabolic stresses, including ketogenic and high-carbohydrate diets, that regulates energy homeostasis. In humans, SNPs in and around the FGF21 gene have been associated with macronutrient preference, including carbohydrate, fat, and protein intake. Here we show that FGF21 administration markedly reduces sweet and alcohol preference in mice and sweet preference in cynomolgus monkeys. In mice, these effects require the FGF21 co-receptor β-Klotho in the central nervous system and correlate with reductions in dopamine concentrations in the nucleus accumbens. Since analogs of FGF21 are currently undergoing clinical evaluation for the treatment of obesity and type 2 diabetes, our findings raise the possibility that FGF21 administration could affect nutrient preference and other reward behaviors in humans.
