Moving Domain Computational Fluid Dynamics to Interface with an Embryonic Model of Cardiac Morphogenesis

Peristaltic contraction of the embryonic heart tube produces time- and spatial-varying wall shear stress (WSS) and pressure gradients (∇P) across the atrioventricular (AV) canal. Zebrafish (Danio rerio) are a genetically tractable system to investigate cardiac morphogenesis. The use of Tg(fli1a:EGFP)^y1 transgenic embryos allowed for delineation and two-dimensional reconstruction of the endocardium. This time-varying wall motion was then prescribed in a two-dimensional moving domain computational fluid dynamics (CFD) model, providing new insights into spatial and temporal variations in WSS and ∇P during cardiac development. The CFD simulations were validated with particle image velocimetry (PIV) across the atrioventricular (AV) canal, revealing an increase in both velocities and heart rates, but a decrease in the duration of atrial systole from early to later stages. At 20-30 hours post fertilization (hpf), simulation results revealed bidirectional WSS across the AV canal in the heart tube in response to peristaltic motion of the wall. At 40-50 hpf, the tube structure undergoes cardiac looping, accompanied by a nearly 3-fold increase in WSS magnitude. At 110-120 hpf, distinct AV valve, atrium, ventricle, and bulbus arteriosus form, accompanied by incremental increases in both WSS magnitude and ∇P, but a decrease in bi-directional flow. Laminar flow develops across the AV canal at 20-30 hpf, and persists at 110-120 hpf. Reynolds numbers at the AV canal increase from 0.07±0.03 at 20-30 hpf to 0.23±0.07 at 110-120 hpf (p< 0.05, n=6), whereas Womersley numbers remain relatively unchanged from 0.11 to 0.13. Our moving domain simulations highlights hemodynamic changes in relation to cardiac morphogenesis; thereby, providing a 2-D quantitative approach to complement imaging analysis.     

Pierce's Disease of Grapevines in Taiwan: Isolation,Cultivation and Pathogenicity of Xylella fastidiosa

Characteristic symptoms of Pierce's disease (PD) in grapevines (Vitis vinifera L.) were observed in 2002 in the major grape production fields of central Taiwan. Disease severity in vineyards varied, and all investigated grape cultivars were affected. Diseased tissues were collected from fields for subsequent isolation and characterization of the causal agent of the disease (Xylella fastidiosa). Koch's postulates were fulfilled by artificially inoculating two purified PD bacteria to grape cultivars Kyoho, Honey Red and Golden Muscat. The inoculated plants developed typical leaf‐scorching symptoms, and similar disease severity developed in the three cultivars from which the bacterium was readily re‐isolated, proving that the leaf scorch of grapevines in Taiwan is caused by the fastidious X. fastidiosa. This confirmed PD of grapevines is also the first report from the Asian Continent. Phylogenetic analyses were performed by comparing the 16S rRNA gene and 16S‐23S rRNA internal transcribed spacer region (16S‐23S ITS) of 12 PD strains from Taiwan with the sequences of 13 X. fastidiosa strains from different hosts and different geographical areas. Results showed that the PD strains of Taiwan were closely related to the American X. fastidiosa grape strains but not to the pear strains of Taiwan, suggesting that the X. fastidiosa grape and pear strains of Taiwan may have evolved independently from each other.     

Some observations on the effect of Daflon (micronized purified flavonoid fraction of Rutaceae aurantiae) in bancroftian filarial lymphoedema

BACKGROUND: Morbidity management is a core component of the global programme for the elimination of lymphatic filariasis. In a double-blind clinical trial, the tolerability and efficacy of Daflon (500 mg) + DEC (25 mg) or DEC (25 mg) alone, twice daily for 90 days, was studied in 26 patients with bancroftian filarial lymphoedema. RESULTS: None of the patients in either drug group reported any adverse reaction throughout the treatment period (90 days). Haematological and biochemical parameters were within normal limits and there was no significant difference between the pre-treatment (day 0) and post-treatment (day 90) values. The group receiving Daflon showed significant reduction in oedema volume from day 90 (140.6 PlusMinus; 18.8 ml) to day 360 (71.8 PlusMinus; 20.7 ml) compared to the pre-treatment (day 0, 198.4 PlusMinus; 16.5 ml) value. This accounted for a 63.8% reduction in oedema volume by day 360 (considering the pre-treatment (day 0) as 100%). In the DEC group, the changes in oedema volume (between day 1 and day 360) were not significant when compared to the pre-treatment (day 0) value. The percentage reduction at day 360 was only 9%, which was not significant (P > 0.05). CONCLUSION: This study has shown that Daflon (500 mg, twice a day for 90 days) is both safe and efficacious in reducing oedema volume in bancroftian filarial lymphoedema. Further clinical trials are essential for strengthening the evidence base on the role of this drug in the morbidity management of lymphatic filariasis.     

The atypical Rho GTPase,RhoU, regulates cell-adhesion molecules during cardiac morphogenesis

The vertebrate heart undergoes early complex morphologic events in order to develop key cardiac structures that regulate its overall function (Fahed et al., 2013). Although many genetic factors that participate in patterning the heart have been elucidated (Tu and Chi, 2012), the cellular events that drive cardiac morphogenesis have been less clear. From a chemical genetic screen to identify cellular pathways that control cardiac morphogenesis in zebrafish, we observed that inhibition of the Rho signaling pathways resulted in failure to form the atrioventricular canal and loop the linear heart tube. To identify specific Rho proteins that may regulate this process, we analyzed cardiac expression profiling data and discovered that RhoU was expressed at the atrioventricular canal during the time when it forms. Loss of RhoU function recapitulated the atrioventricular canal and cardiac looping defects observed in the ROCK inhibitor treated zebrafish. Similar to its family member RhoV/Chp (Tay et al., 2010), we discovered that RhoU regulates the cell junctions between cardiomyocytes through the Arhgef7b/Pak kinase pathway in order to guide atrioventricular canal development and cardiac looping. Inhibition of this pathway resulted in similar underlying cardiac defects and conversely, overexpression of a PAK kinase was able to rescue the loss of RhoU cardiac defect. Finally, we found that Wnt signaling, which has been implicated in atrioventricular canal development (Verhoeven et al., 2011), may regulate the expression of RhoU at the atrioventricular canal. Overall, these findings reveal a cardiac developmental pathway involving RhoU/Arhgef7b/Pak signaling, which helps coordinate cell junction formation between atrioventricular cardiomyocytes to promote cell adhesiveness and cell shapes during cardiac morphogenesis. Failure to properly form these cell adhesions during cardiac development may lead to structural heart defects and mechanistically account for the cellular events that occur in certain human congenital heart diseases.     

A hybrid of light-field and light-sheet imaging to study myocardial function and intracardiac blood flow during zebrafish development

Biomechanical forces intimately contribute to cardiac morphogenesis. However, volumetric imaging to investigate the cardiac mechanics with high temporal and spatial resolution remains an imaging challenge. We hereby integrated light-field microscopy (LFM) with light-sheet fluorescence microscopy (LSFM), coupled with a retrospective gating method, to simultaneously access myocardial contraction and intracardiac blood flow at 200 volumes per second. While LSFM allows for the reconstruction of the myocardial function, LFM enables instantaneous acquisition of the intracardiac blood cells traversing across the valves. We further adopted deformable image registration to quantify the ventricular wall displacement and particle tracking velocimetry to monitor intracardiac blood flow. The integration of LFM and LSFM enabled the time-dependent tracking of the individual blood cells and the differential rates of segmental wall displacement during a cardiac cycle. Taken together, we demonstrated a hybrid system, coupled with our image analysis pipeline, to simultaneously capture the myocardial wall motion with intracardiac blood flow during cardiac development.     

LDL protein nitration: implication for LDL protein unfolding

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL⁻) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL⁻ assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC).The plasma LDL⁻ fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL⁻ revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the α-helical structures and β₂ sheet as well as cysteine oxidation to cysteic acid in β₁ sheet. Circular dichroism analyses showed that the α-helical content of LDL⁻ was substantially lower (∼25%) than that of native LDL (∼90%); conversely, LDL⁻ showed greater content of β-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO⁻) or SIN-1: similar amino acid modifications as well as conformational changes (loss of α-helical structure and gain in β-sheet structure) were observed. Both LDL⁻ and ONOO⁻-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO⁻-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R.It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.     

Sampling properties of DNA sequence data in phylogenetic analysis

We inferred phylogenetic trees from individual genes and random samples of nucleotides from the mitochondrial genomes of 10 vertebrates and compared the results to those obtained by analyzing the whole genomes. Individual genes are poor samples in that they infrequently lead to the whole-genome tree. A large number of nucleotide sites is needed to exactly determine the whole-genome tree. A relatively small number of sites, however, often results in a tree close to the whole-genome tree. We found that blocks of contiguous sites were less likely to lead to the whole-genome tree than samples composed of sites drawn individually from throughout the genome. Samples of contiguous sites are not representative of the entire genome, a condition that violates a basic assumption of the bootstrap method as it is applied in phylogenetic studies.