In Vivo Bypass of 8-oxodG

8-oxoG is one of the most common and mutagenic DNA base lesions caused by oxidative damage. However, it has not been possible to study the replication of a known 8-oxoG base in vivo in order to determine the accuracy of its replication, the influence of various components on that accuracy, and the extent to which an 8-oxoG might present a barrier to replication. We have been able to place a single 8-oxoG into the Saccharomyces cerevisiae chromosome in a defined location using single-strand oligonucleotide transformation and to study its replication in a fully normal chromosome context. During replication, 8-oxoG is recognized as a lesion and triggers a switch to translesion synthesis by Pol η, which replicates 8-oxoG with an accuracy (insertion of a C opposite the 8-oxoG) of approximately 94%. In the absence of Pol η, template switching to the newly synthesized sister chromatid is observed at least one third of the time; replication of the 8-oxoG in the absence of Pol η is less than 40% accurate. The mismatch repair (MMR) system plays an important role in 8-oxoG replication. Template switching is blocked by MMR and replication accuracy even in the absence of Pol η is approximately 95% when MMR is active. These findings indicate that in light of the overlapping mechanisms by which errors in 8-oxoG replication can be avoided in the cell, the mutagenic threat of 8-oxoG is due more to its abundance than the effect of a single lesion. In addition, the methods used here should be applicable to the study of any lesion that can be stably incorporated into synthetic oligonucleotides.     

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest follows every 10 contractions with a total of 30 contractions performed.Download video file.^{(57M, mov)}     

Ex Vivo Effect of Varespladib on Secretory Phospholipase A2 Alveolar Activity in Infants with ARDS

Background

Secretory phospholipase A2 (sPLA2) plays a pivotal role in acute respiratory distress syndrome (ARDS). This enzyme seems an interesting target to reduce surfactant catabolism and lung tissue inflammation. Varespladib is a specifically designed indolic sPLA2 inhibitor, which has shown promising results in animals and adults. No specific data in pediatric ARDS patients are yet available.

Methods

We studied varespladib in broncho-alveolar lavage (BAL) fluids obtained ex vivo from pediatric ARDS patients. Clinical data and worst gas exchange values during the ARDS course were recorded. Samples were treated with saline or 10–40–100 µM varespladib and incubated at 37°C. Total sPLA2 activity was measured by non-radioactive method. BAL samples were subjected to western blotting to identify the main sPLA isotypes with different sensitivity to varespladib. Results was corrected for lavage dilution using the serum-to-BAL urea ratio and for varespladib absorbance.

Results

Varespladib reduces sPLA2 activity (p<0.0001) at 10,40 and 100 µM; both sPLA2 activity reduction and its ratio to total proteins significantly raise with increasing varespladib concentrations (p<0.001). IC₅₀ was 80 µM. Western blotting revealed the presence of sPLA2-IIA and –IB isotypes in BAL samples. Significant correlations exist between the sPLA2 activity reduction/proteins ratio and PaO₂ (rho = 0.63;p<0.001), PaO₂/FiO₂ (rho = 0.7; p<0.001), oxygenation (rho = −0.6; p<0.001) and ventilation (rho = −0.4;p = 0.038) indexes.

Conclusions

Varespladib significantly inhibits sPLA2 in BAL of infants affected by post-neonatal ARDS. Inhibition seems to be inversely related to the severity of gas exchange impairment.     

In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.     

In Vitro and In Vivo Human Herpesvirus 8 Infection of Placenta

Herpesvirus infection of placenta may be harmful in pregnancy leading to disorders in fetal growth, premature delivery, miscarriage, or major congenital abnormalities. Although a correlation between human herpesvirus 8 (HHV-8) infection and abortion or low birth weight in children has been suggested, and rare cases of in utero or perinatal HHV-8 transmission have been documented, no direct evidence of HHV-8 infection of placenta has yet been reported. The aim of this study was to evaluate the in vitro and in vivo susceptibility of placental cells to HHV-8 infection. Short-term infection assays were performed on placental chorionic villi isolated from term placentae. Qualitative and quantitative HHV-8 detection were performed by PCR and real-time PCR, and HHV-8 proteins were analyzed by immunohistochemistry. Term placenta samples from HHV-8-seropositive women were analyzed for the presence of HHV-8 DNA and antigens. In vitro infected histocultures showed increasing amounts of HHV-8 DNA in tissues and supernatants; cyto- and syncitiotrophoblasts, as well as endothelial cells, expressed latent and lytic viral antigens. Increased apoptotic phenomena were visualized by the terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end-labeling method in infected histocultures. Ex vivo, HHV-8 DNA and a latent viral antigen were detected in placenta samples from HHV-8-seropositive women. These findings demonstrate that HHV-8, like other human herpesviruses, may infect placental cells in vitro and in vivo, thus providing evidence that this phenomenon might influence vertical transmission and pregnancy outcome in HHV-8-infected women.     

In Silico Prediction and In Vivo Validation of Daphnia pulex Micrornas

Daphnia pulex, the crustacean with the first sequenced genome, is an important organism that has been widely used in ecological and toxicological research. MicroRNAs (miRNAs) are 21–25 nucleotide small non-coding RNAs that are involved in a myriad of physiological processes. In this research, we predicted 75 D. pulex miRNAs by sequence homology and secondary structure identification from the full genome sequence. Fourteen predicted miRNAs were selected for quantitative real time polymerase chain reaction (RT-PCR) validation. Out of these, eight (mir-8, mir-9, mir-12, mir-92, mir-100, mir-133, mir-153 and mir-283) were successfully amplified and validated. Next, expression levels were quantified at three different life stages (days 4, 8 and 12 of age) using U6 spliceosomal RNA as a reference gene. The expression of mir-8, mir-9, mir-12, mir-92 and mir-100 significantly differed across time suggesting these microRNAs might play a critical role during D. pulex development. This is the first study to identify and validate miRNAs in D. pulex, which is an important first step in further studies that evaluate their roles in development and response to environmental and ecological stimuli.     

In Vivo and In Vitro Characterization of a Plasmodium Liver Stage-Specific Promoter

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems.     

In Vivo Mutational Characterization of DndE Involved in DNA Phosphorothioate Modification

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and R _P stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.     

In Vivo Ultrasonic Detection of Polyurea Crosslinked Silica Aerogel Implants

Background

Polyurea crosslinked silica aerogels are highly porous, lightweight, and mechanically strong materials with great potential for in vivo applications. Recent in vivo and in vitro studies have demonstrated the biocompatibility of this type of aerogel. The highly porous nature of aerogels allows for exceptional thermal, electric, and acoustic insulating capabilities that can be taken advantage of for non-invasive external imaging techniques. Sound-based detection of implants is a low cost, non-invasive, portable, and rapid technique that is routinely used and readily available in major clinics and hospitals.

Methodology

In this study the first in vivo ultrasound response of polyurea crosslinked silica aerogel implants was investigated by means of a GE Medical Systems LogiQe diagnostic ultrasound machine with a linear array probe. Aerogel samples were inserted subcutaneously and sub-muscularly in a) fresh animal model and b) cadaveric human model for analysis. For comparison, samples of polydimethylsiloxane (PDMS) were also imaged under similar conditions as the aerogel samples.

Conclusion/significance

Polyurea crosslinked silica aerogel (X-Si aerogel) implants were easily identified when inserted in either of the regions in both fresh animal model and cadaveric model. The implant dimensions inferred from the images matched the actual size of the implants and no apparent damage was sustained by the X-Si aerogel implants as a result of the ultrasonic imaging process. The aerogel implants demonstrated hyperechoic behavior and significant posterior shadowing. Results obtained were compared with images acquired from the PDMS implants inserted at the same location.     

In Vivo Image Analysis of BoHV-4-Based Vector in Mice

Due to its biological characteristics bovine herpesvirus 4 (BoHV-4) has been considered as an appropriate gene delivery vector. Its genomic clone, modified as a bacterial artificial chromosome (BAC), is better genetically manipulable and can be used as an efficient gene delivery and vaccine vector. Although a large amount of data have been accumulated in vitro on this specific aspect, the same cannot be asserted for the in vivo condition. Therefore, here we investigated the fate of a recombinant BoHV-4 strain expressing luciferase (BoHV-4-A-CMVlucΔTK) after intraperitoneal or intravenous inoculation in mice, by generating a novel recombinant BoHV-4 expressing luciferase (BoHV-4-A-CMVlucΔTK) and by following the virus replication through in vivo imaging analysis. BoHV-4-A-CMVlucΔTK was first characterized in vitro where it was shown, on one hand that its replication properties are identical to those of the parental virus, and on the other that the transduced/infected cells strongly express luciferase. When BoHV-4-A-CMVlucΔTK was inoculated in mice, either intraperitoneally or intravenously, BoHV-4-A-CMVlucΔTK infection/transduction was exclusively localized to the liver, as detected by in vivo image analysis, and in particular almost exclusively in the hepatocytes, as determined by immuno-histochemistry. These data, that add a new insight on the biology of BoHV-4 in vivo, provide the first indication for the potential use of a BoHV-4-based vector in gene-transfer in the liver.     

In Vivo Analysis of the Notch Receptor S1 Cleavage

A ligand-independent cleavage (S1) in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.     

In Vivo Volatile Organic Compound Signatures of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of a chronic enteric disease of ruminants. Available diagnostic tests are complex and slow. In vitro, volatile organic compound (VOC) patterns emitted from MAP cultures mirrored bacterial growth and enabled distinction of different strains. This study was intended to determine VOCs in vivo in the controlled setting of an animal model. VOCs were pre-concentrated from breath and feces of 42 goats (16 controls and 26 MAP-inoculated animals) by means of needle trap microextraction (breath) and solid phase microextraction (feces) and analyzed by gas chromatography/ mass spectrometry. Analyses were performed 18, 29, 33, 41 and 48 weeks after inoculation. MAP-specific antibodies and MAP-specific interferon-γ-response were determined from blood. Identities of all marker-VOCs were confirmed through analysis of pure reference substances. Based on detection limits in the high pptV and linear ranges of two orders of magnitude more than 100 VOCs could be detected in breath and in headspace over feces. Twenty eight substances differed between inoculated and non-inoculated animals. Although patterns of most prominent substances such as furans, oxygenated substances and hydrocarbons changed in the course of infection, differences between inoculated and non-inoculated animals remained detectable at any time for 16 substances in feces and 3 VOCs in breath. Differences of VOC concentrations over feces reflected presence of MAP bacteria. Differences in VOC profiles from breath were linked to the host response in terms of interferon-γ-response. In a perspective in vivo analysis of VOCs may help to overcome limitations of established tests.     

In Vitro and In Vivo Activity of an Organic Tellurium Compound on Leishmania (Leishmania) chagasi

Tellurium compounds have shown several biological properties and recently the leishmanicidal effect of one organotellurane was demonstrated. These findings led us to test the effect of the organotellurium compound RF07 on Leishmania (Leishmania) chagasi, the agent of visceral leishmaniasis in Latin America. In vitro assays were performed in L. (L.) chagasi-infected bone marrow derived macrophages treated with different concentrations of RF07. In in vivo experiments Golden hamsters were infected with L. (L.) chagasi and injected intraperitoneally with RF07 whereas control animals received either Glucantime or PBS. The effect of RF07 on cathepsin B activity of L. (L.) chagasi amastigotes was assayed spectrofluorometrically using fluorogenic substrates. The main findings were: 1) RF07 showed significant leishmanicidal activity against intracellular parasites at submicromolar concentrations (IC50 of 529.7±26.5 nM), and the drug displayed 10-fold less toxicity to macrophages (CC50 of 5,426±272.8 nM); 2) kinetics assays showed an increasing leishmanicidal action of RF07 at longer periods of treatment; 3) one month after intraperitoneal injection of RF07 L. (L.) chagasi-infected hamsters showed a reduction of 99.6% of parasite burden when compared to controls that received PBS; 4) RF07 inhibited the cathepsin B activity of L. (L.) chagasi amastigotes. The present results demonstrated that the tellurium compound RF07 is able to destroy L. (L.) chagasi in vitro and in vivo at concentrations that are non toxic to the host. We believe these findings support further study of the potential of RF07 as a possible alternative for the chemotherapy of visceral leishmaniasis.     

In Vivo RNAi Rescue in Drosophila melanogaster with Genomic Transgenes from Drosophila pseudoobscura

Background

Systematic, large-scale RNA interference (RNAi) approaches are very valuable to systematically investigate biological processes in cell culture or in tissues of organisms such as Drosophila. A notorious pitfall of all RNAi technologies are potential false positives caused by unspecific knock-down of genes other than the intended target gene. The ultimate proof for RNAi specificity is a rescue by a construct immune to RNAi, typically originating from a related species.

Methodology/Principal Findings

We show that primary sequence divergence in areas targeted by Drosophila melanogaster RNAi hairpins in five non-melanogaster species is sufficient to identify orthologs for 81% of the genes that are predicted to be RNAi refractory. We use clones from a genomic fosmid library of Drosophila pseudoobscura to demonstrate the rescue of RNAi phenotypes in Drosophila melanogaster muscles. Four out of five fosmid clones we tested harbour cross-species functionality for the gene assayed, and three out of the four rescue a RNAi phenotype in Drosophila melanogaster.

Conclusions/Significance

The Drosophila pseudoobscura fosmid library is designed for seamless cross-species transgenesis and can be readily used to demonstrate specificity of RNAi phenotypes in a systematic manner.     

In Vivo Quantitative Susceptibility Mapping (QSM) in Alzheimer's Disease

Background

This study explores the magnetostatic properties of the Alzheimer''s disease brain using a recently proposed, magnetic resonance imaging, postprocessed contrast mechanism. Quantitative susceptibility mapping (QSM) has the potential to monitor in vivo iron levels by reconstructing magnetic susceptibility sources from field perturbations. However, with phase data acquired at a single head orientation, the technique relies on several theoretical approximations and requires fast-evolving regularisation strategies.

Methods

In this context, the present study describes a complete methodological framework for magnetic susceptibility measurements with a review of its theoretical foundations.

Findings and Significance

The regional and whole-brain cross-sectional comparisons between Alzheimer''s disease subjects and matched controls indicate that there may be significant magnetic susceptibility differences for deep brain nuclei – particularly the putamen – as well as for posterior grey and white matter regions. The methodology and findings described suggest that the QSM method is ready for larger-scale clinical studies.     

In Vivo Wall Shear Measurements within the Developing Zebrafish Heart

Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.