New model for in vivo investigation after microvascular breakdown in burns: use of intravital fluorescent microscopy

AbstractMicrobiota within mosquitoes influence nutrition, immunity, fecundity, and the capacity to transmit pathogens. Despite their importance, we have a limited understanding of host-microbiota interactions, especially at the cellular level. It is evident bacterial symbionts that are localized within the midgut also infect other organs within the mosquito; however, the route these symbionts take to colonize other tissues is unknown. Here, utilizing the gentamicin protection assay, we showed that the bacterial symbionts Cedecea and Serratia have the capacity to invade and reside intracellularly within mosquito cells. Symbiotic bacteria were found within a vacuole and bacterial replication was observed in mosquito cell by transmission electron microscopy, indicating bacteria were adapted to the intracellular milieu. Using gene silencing, we determined that bacteria exploited host factors, including actin and integrin receptors, to actively invade mosquito cells. As microbiota can affect pathogens within mosquitoes, we examined the influence of intracellular symbionts on Zika virus (ZIKV) infection. Mosquito cells harbouring intracellular bacteria had significantly less ZIKV compared to uninfected cells or cells exposed to non-invasive bacteria. Intracellular bacteria were observed to substantially upregulate the Toll and IMD innate immune pathways, providing a possible mechanism mediating these anti-viral effects. Examining mono-axenically infected mosquitoes using transmission electron and fluorescent microscopy revealed that bacteria occupied an intracellular niche in vivo. Our results provided evidence that bacteria that associate with the midgut of mosquitoes have intracellular lifestyles which likely have implications for mosquito biology and pathogen infection. This study expands our understanding of host-microbiota interactions in mosquitoes, which is important as symbiont microbes are being exploited for vector control strategies.

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The three dimensional structure of Bovine Salivary Protein 30b (BSP30b) and its interaction with specific rumen bacteria

10.1101/448167 ◽

2018 ◽

Author(s):

Heng Zhang ◽

Judith Burrows ◽

Graeme L. Card ◽

Graeme Attwood ◽

Thomas T. Wheeler ◽

...

Keyword(s):

Oleic Acid ◽

Binding Proteins ◽

Bacterial Species ◽

Fluorescent Microscopy ◽

Salivary Protein ◽

Lipid Binding ◽

Dimensional Structure ◽

Rumen Bacteria ◽

Abundant Protein

AbstractBovine Salivary Protein 30b (BSP30b) is a member of the tubular lipid-binding (TULIP) superfamily that includes the human bactericidal/permeability-increasing proteins (BPI), lipopolysaccharide binding proteins (LBP) and palate, lung, and nasal epithelium carcinoma-21 associated proteins (PLUNC). BSP30b is most closely related to the PLUNC family and is predominantly found in bovine saliva. There are four BSP30 isoforms (BSP30a-d) and collectively, they are the most abundant protein component of bovine saliva. The PLUNC family members are proposed to be lipid binding proteins, although in most cases their lipid ligands are unknown. Here, we present the X-ray crystal structure of BSP30b at 2.0 Å resolution. We used a double methionine mutant and Se-Met SAD phasing to solve the structure. The structure adopts a curved cylindrical form with a hydrophobic channel formed by an α/β wrap, which is consistent with the TULIP superfamily. The structure of BSP30b in complex with oleic acid is also presented where the ligand is accommodated within the hydrophobic channel. The electron density for oleic acid suggests that the ligand is only partially occupied in the binding site implying that oleic acid may not be the preferred ligand. GFP-tagged BSP30b binds to the surface of olive oil droplets, as observed under fluorescent microscopy, and acts as a surfactant consistent with its association with decreased susceptibility to bloat in cattle. Bacteria extracted directly from bovine rumen contents indicate that the GFP_BSP30b fusion protein binds to a small number of selected bacterial species in vivo. These results suggest that BSP30b may bind to bacterial lipids from specific species and that this abundant protein may have important biological roles via interacting with rumen bacteria during feeding and rumination.

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Monodelphis domestica as a fetal intra-cerebral inoculation model for Zika virus pathogenesis

10.1101/785220 ◽

2019 ◽

Author(s):

John M. Thomas ◽

Juan Garcia ◽

Matthew Terry ◽

Ileana Lozano ◽

Susan M. Mahaney ◽

...

Keyword(s):

Animal Model ◽

Zika Virus ◽

Developmental Stages ◽

Experimental Manipulation ◽

Monodelphis Domestica ◽

Growth Restriction ◽

Human Embryos ◽

New Model ◽

Global Growth

ABSTRACTMonodelphis domestica, also known as the laboratory opossum, is a marsupial native to South America. At birth, these animals are developmentally equivalent to human embryos at approximately 5 weeks of gestation which, when coupled with other characteristics including the size of the animals, the development of a robust immune system during juvenile development, and the relative ease of experimental manipulation, have made M. domestica a valuable model in many areas of biomedical research. However, their suitability as models for infectious diseases, especially diseases caused by viruses such as Zika virus (ZIKV), is currently unknown. Here, we describe the replicative effects of ZIKV using a fetal intra-cerebral model of inoculation. Using immunohistochemistry and in situ hybridization, we found that opossum embryos and fetuses are susceptible to infection by ZIKV administered intra-cerebrally, that the infection persists long term, and that the infection and viral replication consistently results in neural pathology and may occasionally result in global growth restriction. These results demonstrate the utility of M. domestica as a new animal model for investigating ZIKV infection in vivo. This new model will facilitate further inquiry into viral pathogenesis, particularly for those viruses that are neurotropic, that may require a host with the ability to support sustained viral infection, and/or that may require intra-cerebral inoculations of large numbers of embryos or fetuses.AUTHOR SUMMARYHere we show that the laboratory opossum (Monodelphis domestica) is a valuable new model for studying Zika virus pathogenesis. Newborns are at the developmental stage of 5-week human embryos. Zika virus inoculated on a single occasion into the brains of pups at the human developmental stages of 8-20 weeks post conception replicated in neuronal cells and persisted as a chronic infection until the experimental endpoint at 74-days post infection. In addition, we observed global growth restriction in one of 16 inoculated animals; global growth restriction has been observed in humans and other animal models infected with Zika virus. The results illustrate great potential for this new animal model for high throughput research on the neurological effects of Zika virus infection of embryos and fetuses.

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3D visualization of macromolecule synthesis

eLife ◽

10.7554/elife.60354 ◽

2020 ◽

Vol 9 ◽

Author(s):

Timothy J Duerr ◽

Ester Comellas ◽

Eun Kyung Jeon ◽

Johanna E Farkas ◽

Marylou Joetzjer ◽

...

Keyword(s):

3D Visualization ◽

Fluorescent Microscopy ◽

Three Dimensional ◽

Light Sheet ◽

Macromolecular Synthesis ◽

Volumetric Imaging ◽

Processing Pipeline ◽

Powerful Means

Measuring nascent macromolecular synthesis in vivo is key to understanding how cells and tissues progress through development and respond to external cues. Here we perform in vivo injection of alkyne- or azide-modified analogs of thymidine, uridine, methionine, and glucosamine to label nascent synthesis of DNA, RNA, protein, and glycosylation. Three-dimensional volumetric imaging of nascent macromolecule synthesis was performed in axolotl salamander tissue using whole-mount click chemistry-based fluorescent staining followed by light sheet fluorescent microscopy. We also developed an image processing pipeline for segmentation and classification of morphological regions of interest and individual cells, and we apply this pipeline to the regenerating humerus. We demonstrate our approach is sensitive to biological perturbations by measuring changes in DNA synthesis after limb denervation. This method provides a powerful means to quantitatively interrogate macromolecule synthesis in heterogenous tissues at the organ, cellular, and molecular levels of organization.

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Cancer formation in WAP-HBV-X transgenic animals a new model to study pX oncogenic potential in vivo

Hepatology ◽

10.1016/0270-9139(95)94499-0 ◽

1995 ◽

Vol 22 (4) ◽

pp. A194

Author(s):

M LEVRERO

Keyword(s):

Transgenic Animals ◽

Oncogenic Potential ◽

New Model

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Leukocyte-Endothelium Interactions during Permanent Focal Cerebral Ischemia in Mice

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000117812.35136.5b ◽

2004 ◽

Vol 24 (6) ◽

pp. 668-676 ◽

Cited By ~ 41

Author(s):

Hiroharu Kataoka ◽

Seong-Woong Kim ◽

Nikolaus Plesnila

Keyword(s):

Cerebral Ischemia ◽

Brain Damage ◽

Molecular Mechanisms ◽

Focal Cerebral Ischemia ◽

Fluorescent Microscopy ◽

Capillary Density ◽

Artery Occlusion ◽

Cerebral Artery Occlusion ◽

Adherent Leukocytes

The contribution of leukocyte infiltration to brain damage after permanent focal cerebral ischemia and the underlying molecular mechanisms are still unclear. Therefore, the aim of this study was to establish a mouse model for the visualization of leukocytes in the cerebral microcirculation in vivo and to investigate leukocyte-endothelial interaction (LEI) after permanent middle cerebral artery occlusion (MCAO). Sham-operated 129/Sv mice showed physiologic LEI in pial venules as observed by intravital fluorescent microscopy. Permanent focal cerebral ischemia induced a significant increase of LEI predominantly in pial venules. The number of rolling and adherent leukocytes reached 36.5 ± 13.2/100 μm × min and 22.5 ± 7.9/100 μm × min, respectively at 120 minutes after MCAO ( P = 0.016 vs. control). Of note, rolling and adherent leukocytes were also observed in arterioles of ischemic animals (7.3 ± 3.0/100 μm × min rolling and 3.0 ± 3.6/100 μm × min adherent). Capillary density was not different between groups. These results demonstrate that leukocytes accumulate in the brain not only after transient but also after permanent focal cerebral ischemia and may therefore contribute to brain damage after stroke without reperfusion.

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