Searching for the Optimal Fluorophore to Label Antimicrobial Peptides

With the advent of antimicrobial resistance, there is an urgent need for new strategies to treat infectious diseases. Antimicrobial peptides are considered as promising candidates, and therefore there is a need to understand their mechanism of action in order to exploit their therapeutic potential. To this end, fluorescent analogs are powerful tools to analyze their behavior and subcellular localization in cells and in vivo. However, the conjugation of fluorophores to antimicrobial peptides, especially in short sequences, can impair their biological activity, making the selection of the fluorescent label an essential step in these studies. In the present work, we have systematically modified a model antifungal hexapeptide with a collection of fluorophores covering broad physicochemical and spectral properties. The resulting conjugates have been examined in two different fungal species, in terms of their activity and intracellular localization. The biological results confirm the influence of the different fluorescent moieties on the subcellular localization of antimicrobial sequences, and provides an insight on the optimal fluorophores to be used in the preparation of fluorescent peptides for different bioimaging assays

Lung Volume Calculation in Preclinical MicroCT: A Fast Geometrical Approach

Lung; Preclinical imaging; VolumePulmón; Imágenes preclínicas; VolumenPulmó; Imatges preclíniques; VolumIn this study, we present a time-efficient protocol for thoracic volume calculation as a proxy for total lung volume. We hypothesize that lung volume can be calculated indirectly from this thoracic volume. We compared the measured thoracic volume with manually segmented and automatically thresholded lung volumes, with manual segmentation as the gold standard. A linear regression formula was obtained and used for calculating the theoretical lung volume. This volume was compared with the gold standard volumes. In healthy animals, thoracic volume was 887.45 mm3, manually delineated lung volume 554.33 mm3 and thresholded aerated lung volume 495.38 mm3 on average. Theoretical lung volume was 554.30 mm3. Finally, the protocol was applied to three animal models of lung pathology (lung metastasis and transgenic primary lung tumor and fungal infection). In confirmed pathologic animals, thoracic volumes were: 893.20 mm3, 860.12 and 1027.28 mm3. Manually delineated volumes were 640.58, 503.91 and 882.42 mm3, respectively. Thresholded lung volumes were 315.92 mm3, 408.72 and 236 mm3, respectively. Theoretical lung volume resulted in 635.28, 524.30 and 863.10.42 mm3. No significant differences were observed between volumes. This confirmed the potential use of this protocol for lung volume calculation in pathologic models

Fate of mesoangioblasts in a vaginal birth injury model: influence of the route of administration

Currently cell therapy is considered as an experimental strategy to assist the healing process following simulated vaginal birth injury in rats, boosting the functional and morphologic recovery of pelvic floor muscles and nerves. However, the optimal administration route and dose still need to be determined. Mesangioblasts theoretically have the advantage that they can differentiate in skeletal and smooth muscle. We investigated the fate of mesoangioblasts transduced with luciferase and green fluorescent protein reporter genes (rMABseGFP/fLUC) using bioluminescence, immunofluorescence and RT-PCR in rats undergoing simulated birth injury. rMABseGFP/fLUC were injected locally, intravenously and intra-arterially (common iliacs and aorta). Intra-arterial delivery resulted in the highest amount of rMABseGFP/fLUC in the pelvic organs region and in a more homogeneous distribution over all relevant pelvic organs. Sham controls showed that the presence of the injury is important for recruitment of intra-arterially injected rMABseGFP/fLUC. Injection through the aorta or bilaterally in the common iliac arteries resulted in comparable numbers of rMABseGFP/fLUC in the pelvic organs, yet aortic injection was faster and gave less complications

Green Tea catechins modulate skeletal development with effects dependent on dose, time, and structure in a Down syndrome mouse model

Altered skeletal development in Down syndrome (DS) results in a brachycephalic skull, flattened face, shorter mandibular ramus, shorter limbs, and reduced bone mineral density (BMD). Our previous study showed that low doses of green tea extract enriched in epigallocatechin-3-gallate (GTE-EGCG), administered continuously from embryonic day 9 to postnatal day 29, reduced facial dysmorphologies in the Ts65Dn (TS) mouse model of DS, but high doses could exacerbate them. Here, we extended the analyses to other skeletal structures and systematically evaluated the effects of high and low doses of GTE-EGCG treatment over postnatal development in wild-type (WT) and TS mice using in vivo µCT and geometric morphometrics. TS mice developed shorter and wider faces, skulls, and mandibles, together with shorter and narrower humerus and scapula, and reduced BMD dynamically over time. Besides facial morphology, GTE-EGCG did not rescue any other skeletal phenotype in TS treated mice. In WT mice, GTE-EGCG significantly altered the shape of the skull and mandible, reduced the length and width of the long bones, and lowered the BMD. The disparate effects of GTE-EGCG depended on the dose, developmental timepoint, and anatomical structure analyzed, emphasizing the complex nature of DS and the need to further investigate the simultaneous effects of GTE-EGCG supplementation

Placental vascular alterations are associated with early neurodevelopmental and pulmonary impairment in the rabbit fetal growth restriction model

Fetal growth restriction is one of the leading causes of perinatal mortality and morbidity and has consequences that extend well beyond the neonatal period. Current management relies on timely delivery rather than improving placental function. Several prenatal strategies have failed to show benefit in clinical trials after promising results in animal models. Most of these animal models have important developmental and structural differences compared to the human and/or are insufficiently characterized. We aimed to describe placental function and structure in an FGR rabbit model, and to characterize the early brain and lung developmental morbidity using a multimodal approach. FGR was induced in time-mated rabbits at gestational day 25 by partial uteroplacental vessel ligation in one horn. Umbilical artery Doppler was measured before caesarean delivery at gestational day 30, and placentas were harvested for computed microtomography and histology. Neonates underwent neurobehavioral or pulmonary functional assessment the day after delivery, followed by brain or lung harvesting, respectively. Neuropathological assessment included multiregional quantification of neuron density, apoptosis, astrogliosis, cellular proliferation, and oligodendrocyte progenitors. Brain region volumes and diffusion metrics were obtained from ex-vivo brain magnetic resonance imaging. Lung assessment included biomechanical tests and pulmonary histology. Fetal growth restriction was associated with labyrinth alterations in the placenta, driven by fetal capillary reduction, and overall reduced vessels volume. FGR caused altered neurobehavior paralleled by regional neuropathological deficits and reduced fractional anisotropy in the cortex, white matter, and hippocampus. In addition, FGR kittens presented functional alterations in the peripheral lung and structurally underdeveloped alveoli. In conclusion, in a uteroplacental insufficiency FGR rabbit model, placental vascular alterations coincide with neurodevelopmental and pulmonary disruption

A multimodal imaging approach enables in vivo assessment of antifungal treatment in a mouse model of invasive pulmonary aspergillosis

Aspergillus fumigatus causes life-threatening lung infections in immunocompromised patients. Mouse models are extensively used in research to assess the in vivo efficacy of antifungals. In recent years, there has been an increasing interest in the use of non-invasive imaging techniques to evaluate experimental infections. However, single imaging modalities have limitations concerning the type of information they can provide. In this study, magnetic resonance imaging and bioluminescence imaging were combined to obtain longitudinal information on the extent of developing lesions and fungal load in a leucopenic mouse model of IPA. This multimodal imaging approach was used to assess changes occurring within lungs of infected mice receiving voriconazole treatment starting at different time points after infection. Results showed that IPA development depends on the inoculum size used to infect animals and that disease can be successfully prevented or treated by initiating intervention during early stages of infection. Furthermore, we demonstrated that reduction of the fungal load is not necessarily associated with the disappearance of lesions on anatomical lung images, especially when antifungal treatment coincides with immune recovery. In conclusion, multimodal imaging allows to investigate different aspects of disease progression or recovery by providing complementary information on dynamic processes, which are highly useful for assessing the efficacy of (novel) therapeutic compounds in a time- and labor-efficient manner

Longitudinal in vivo assessment of host-microbe interactions in a murine model of pulmonary aspergillosis

The fungus Aspergillus fumigatus is ubiquitous in nature and the most common cause of invasive pulmonary aspergillosis (IPA) in patients with a compromised immune system. The development of IPA in patients under immunosuppressive treatment or in patients with primary immunodeficiency demonstrates the importance of the host immune response in controlling aspergillosis. However, study of the host-microbe interaction has been hampered by the lack of tools for their non-invasive assessment. We developed a methodology to study the response of the host's immune system against IPA longitudinally in vivo by using fluorine-19 magnetic resonance imaging (F-19 MRI). We showed the advantage of a perfluorocarbon-based contrast agent for the in vivo labeling of macrophages and dendritic cells, permitting quantification of pulmonary inflammation in different murine IPA models. Our findings reveal the potential of F-19 MRI for the assessment of rapid kinetics of innate immune response against IPA and the permissive niche generated through immunosuppression