21 research outputs found
Characterization of aortic root atherosclerosis in ApoE knockout mice: high-resolution in vivo and ex vivo MRM with histological correlation.
No full textIn vivo, cardiac-gated, black-blood, and ex vivo magnetic resonance microscopy (MRM) images of the aortic root, and histopathology data were obtained from 12 transgenic and wild-type (WT) mice. MRM was performed using a black-blood imaging spin-echo sequence with upstream and downstream in-flow saturation pulses to obtain aortic root images in three contrast techniques: proton density-weighted (PDW), T(1)- (T(1)W), and T(2)-weighted (T(2)W). Aortic wall thickness and area were measured and correlated with histopathology data (R > 0.90). Ex vivo lesion components (lipid core, fibrous tissue, and cell tissue) were identified and characterized by differing image contrast in PDW, T(1)W, and T(2)W MRM, and by histopathology. The differences between WT and transgenic mice for maximal wall thickness and area were statistically significant (P < 0.05). This study demonstrates the feasibility of in vivo murine aortic root lesion assessment and ex vivo plaque characterization by MRM
Characterization of functional broad-host range replicons in the honey bee gut symbiont <i>B</i>. <i>apis</i>.
No full text(a) Broad-host range plasmids have different copy numbers in B. apis. Box plots show median values of plasmid copy numbers obtained by qPCR from 3 independent experiments with 5 biological replicate each (total n = 15). Median copy number are indicated with the corresponding box plots. (b) The difference in plasmid copy number results in different protein expression levels in B. apis. Graph shows mean of E2-crimson fluorescence ± standard deviations of 5 biological replicates. Each replicate represents the average fluorescence of at least 9,000 cells measured by flow cytometry. Plasmids used for panels a and b in B. apis were pBTK570, pAC06, pAC11, and pAC04, carrying the RSF1010, RK2, pTF-FC2, and pBBR1 origins of replication, respectively. (c) Some replicons are compatible and can be cotransformed in B. apis. Matrix table indicates compatible (green boxes with check mark) and incompatible (red boxes with cross mark) replicons. Vectors were found compatible upon their successful cotransformation by conjugation in B. apis cells. The data underlying this Figure can be found in the S1 Data file, sheets “Supplementary Fig 4A” and “Supplementary Fig 4B.” (PDF)</p
A novel method for noninvasive sampling of the honey bee gut.
No full text(a) Photograph of a bee feces extraction. (b) Box plot indicates the median volume of extracted feces from n = 18 bees. (c) Dot plot shows bacterial load of engineered S. alvi found in the feces and in the gut homogenates. Dotted lines between data points indicate matching samples (i.e., feces and gut were sourced from the same bee). Number of bees sampled (n) and the rate of colonization (r) for the experiment are indicated. (d) Feces collection does not significantly decrease bees fitness. Kaplan–Meier graph shows honeybee’s probability of survival over time when subjected to fecal extractions (FE-treated; solid line) compared to control bees (Control; dotted line). Times of feces collection are indicated by arrows. Log-rank test, not significant (ns) at p-value = 0.112 for n = 44 bees. The data underlying this Figure can be found in the S1 Data file, sheets “Fig 1B,” “Fig 1C,” and “Fig 1D”.</p
Testing of different IPTG-inducible constructs in <i>S</i>. <i>alvi</i>.
No full text(a) Maps of the IPTG-inducible plasmids built in this study. (b) S. alvi cells engineered with our inducible plasmids respond to IPTG exposure in vitro. Graph shows box plots representing median value of GFP fluorescence of 5 biological replicates for each construct tested. Each replicate value is based on the average fluorescence of at least 9,000 S. alvi cells measured by flow cytometry, which were grown in liquid with (+) or without (−) IPTG. As a reference, wild-type S. alvi, S. alvi bearing the pAC08 plasmid constitutively expressing GFP, and S. alvi carrying the previously built pBTK552 vector [28] were also analyzed. Fold-changes of average fluorescence between uninduced and induced cells are indicated. The data underlying this Figure can be found in the S1 Data file, sheet “Supplementary Fig 6B.” (PDF)</p
Collection of broad-host range plasmids developed in this study.
No full text(a) Schematic showing the different available combinations of origin of replications and standardized fragments bearing the antibiotic marker (ampR, specR) and fluorescent reporter (crim, gfp). (b) The names of the corresponding plasmids are indicated. Detailed plasmid maps can be found in S2 Fig.</p
Fiji macro.
No full textThe honey bee is a powerful model system to probe host–gut microbiota interactions, and an important pollinator species for natural ecosystems and for agriculture. While bacterial biosensors can provide critical insight into the complex interplay occurring between a host and its associated microbiota, the lack of methods to noninvasively sample the gut content, and the limited genetic tools to engineer symbionts, have so far hindered their development in honey bees. Here, we built a versatile molecular tool kit to genetically modify symbionts and reported for the first time in the honey bee a technique to sample their feces. We reprogrammed the native bee gut bacterium Snodgrassella alvi as a biosensor for IPTG, with engineered cells that stably colonize the gut of honey bees and report exposure to the molecules in a dose-dependent manner through the expression of a fluorescent protein. We showed that fluorescence readout can be measured in the gut tissues or noninvasively in the feces. These tools and techniques will enable rapid building of engineered bacteria to answer fundamental questions in host–gut microbiota research.</div
Bacterial load from feces is a proxy for levels of gut colonization.
No full textScatterplot shows linear regression of bacterial concentration values of engineered S. alvi found in matching samples of feces and gut homogenates (i.e., feces and gut were sourced from the same bee). Pearson correlation coefficient R and p-value are provided, for n = 22. The data underlying this Figure can be found in the S1 Data file, sheet “Supplementary Fig 1”. (PDF)</p
Validation of qPCR primers and standard curves.
No full textPrimers specificity was confirmed by visualization of amplicons on an agarose gel (left panel) and generation of melting curves (middle panel). Standard curves were generated using serially diluted genomic DNA of (a) S. alvi, (b) B. apis, or (c) miniprep of pBTK570. The data underlying this Figure can be found in the S1 Data file, sheets “Supplementary Fig 3A,” “Supplementary Fig 3B,” and “Supplementary Fig 3C.” (PDF)</p
Engineered <i>S</i>. <i>alvi</i> sense IPTG in vivo and report exposure in the gut tissue and feces.
No full text(a) Schematic of the IPTG-inducible dual plasmid system pAC17V5. (b) S. alvi cells engineered with pAC17V5 respond to IPTG exposure in vitro. Box plots represent median values of GFP fluorescence of 5 biological replicates. Each replicate value is based on the average fluorescence of at least 9,000 S. alvi cells measured by flow cytometry, which were grown in liquid with (+) or without (−) IPTG. (c) Schematic outline of the in vivo experiment using reprogrammed S. alvi to sense IPTG in situ. (d) Confocal microscopy image of a bee gut colonized with engineered S. alvi cells bearing pAC17V5. A biofilm of S. alvi within a gut crypt is shown. The bee was fed with sugar water supplemented with 1 mM IPTG. Blue channel shows DAPI staining of host and bacterial cells. Green channel shows GFP fluorescence. (e) S. alvi engineered with pAC17V5 display a dose-response to IPTG exposure in vivo, which can be measured from gut tissue. Graph shows box plots representing median value of GFP fluorescence of S. alvi biofilms imaged from the gut of bees fed sugar water supplemented with either 0, 0.1, or 1 mM IPTG. Five bees were analyzed for each condition, and fluorescence values were averaged from 3 distinct sections of each gut. Tukey HSD test, * significant at q-value (f) Confocal microscopy image of bee feces, collected from an individual fed with sugar water supplemented with 1 mM IPTG and colonized with S. alvi cells bearing pAC17V5. Green channel shows GFP fluorescence. (g) S. alvi engineered with pAC17V5 display a dose-response to IPTG exposure in vivo, which can be measured from the feces. Box plots represent median values of GFP fluorescence of individual S. alvi cells imaged from the feces of bees fed sugar water supplemented with either 0, 0.1, or 1 mM IPTG. At least 8 bees were analyzed for each condition. The cumulated number n of cells analyzed per conditions is indicated. Tukey HSD test, * significant at q-value q-value S1 Data file, sheets “Fig 5B,” “Fig 5E,” and “Fig 5G”.</p
Maintenance of broad-host range plasmids and engineered <i>S</i>. <i>alvi</i> over time.
No full textEach broad-host-range vector has a distinct level of maintenance in S. alvi cells, as demonstrated in vitro (a) and in vivo (c). Box plots represent plasmid maintenance median values of n biological replicates. Plasmid maintenance was estimated by measuring the percentage of fluorescent colony forming units (CFUs) plated onto nonselective media of samples obtained from (a) liquid cultures (in vitro) after 3 days of growth with (+Ab) or without (−Ab) spectinomycin supplementation; or (c) from feces (in vivo) collected 7 (orange) and 14 (red) days after gut monocolonization of bees continuously fed sugar water supplemented with (+Ab) or without (−Ab) spectinomycin. Only samples for which we detected bacterial colonies upon plating the fecal material were considered. (b) Schematic outline of the in vivo experiment to estimate engineered S. alvi colonization maintenance. Arrows indicate the 2 times of feces sampling. (d) Bacterial load of engineered S. alvi cells found in feces collected 7 (orange) and 14 (red) days after gut monocolonization of bees continuously fed sugar water supplemented with (+Ab) or without (−Ab) spectinomycin. Concentrations of our different S. alvi strains isolated from the feces of bees co-colonized with the natural gut community and that were fed sugar water without spectinomycin (+Gc) was also analyzed. Those CFUs values were obtained by plating diluted feces onto selective media (i.e., supplemented with spectinomycin). Bees for which we did not detect engineered bacteria were considered noncolonized individuals. The rate of colonized bees (r) and the number of bees sampled (n) are provided. Corresponding median values of colonized bees only are represented by colored horizontal bars with interquartile ranges. Plasmids used for all panels in S. alvi were pAC10, pAC04, pBTK570, and pAC11, carrying the pVS1, pBBR1, RSF1010, and pTF-FC2 origins of replication, respectively. The data underlying this Figure can be found in the S1 Data file, sheets “Fig 4A,” “Fig 4C,” and “Fig 4D”.</p
