The level of BMP4 signaling is critical for the regulation of distinct T-box gene expression domains and growth along the dorso-ventral axis of the optic cup

Background  

Polarised gene expression is thought to lead to the graded distribution of signaling molecules providing a patterning mechanism across the embryonic eye. Bone morphogenetic protein 4 (Bmp4) is expressed in the dorsal optic vesicle as it transforms into the optic cup. Bmp4 deletions in human and mouse result in failure of eye development, but little attempt has been made to investigate mammalian targets of BMP4 signaling. In chick, retroviral gene overexpression studies indicate that Bmp4 activates the dorsally expressed Tbx5 gene, which represses ventrally expressed cVax. It is not known whether the Tbx5 related genes, Tbx2 and Tbx3, are BMP4 targets in the mammalian retina and whether BMP4 acts at a distance from its site of expression. Although it is established that Drosophila Dpp (homologue of vertebrate Bmp4) acts as a morphogen, there is little evidence that BMP4 gradients are interpreted to create domains of BMP4 target gene expression in the mouse.     

Nest desertion by a cowbird host: an antiparasite behavior or a response to egg loss?

Natural selection can favor songbirds that desert nests containingeggs of the parasitic brown-headed cowbird (Molothrus ater).However, the high variability in desertion of parasitized nestswithin species is perplexing in light of the typically highcosts of parasitism. Because nest desertion can also be a responseto partial clutch predation, we first asked if Bell's vireos(Vireo bellii) deserted nests in response to the presence ofcowbird eggs (antiparasite response hypothesis) or to egg removalby predators and female cowbirds (egg predation hypothesis).Second, we asked whether variation in nest desertion was dueto intrinsic differences among individuals or to variation innest contents. We monitored a large number of nests (n = 494)and performed a clutch manipulation experiment to test thesehypotheses. The number of vireo eggs that remained in a nestwas a strong predictor of desertion both within and among pairs.Neither the presence of a single cowbird egg, which leads tonest failure for this host, nor the number of cowbird eggs receivedin a vireo nest influenced nest desertion. Furthermore, vireosdid not desert experimental nests when we immediately exchangedcowbird eggs for vireo eggs but deserted if we removed vireoeggs and replaced them with cowbird eggs the following morning.Desertion of parasitized nests by Bell's vireos can be almostentirely explained as a response to partial or complete clutchloss and does not appear to have been altered by selection frombrood parasitism.     

A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis

Introduction

Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.

Methods

Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.

Results

By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.

Conclusions

Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.     

Effect of calcification on the mechanical stability of plaque based on a three-dimensional carotid bifurcation model

Background

This study characterizes the distribution and components of plaque structure by presenting a three-dimensional blood-vessel modelling with the aim of determining mechanical properties due to the effect of lipid core and calcification within a plaque. Numerical simulation has been used to answer how cap thickness and calcium distribution in lipids influence the biomechanical stress on the plaque.

Method

Modelling atherosclerotic plaque based on structural analysis confirms the rationale for plaque mechanical examination and the feasibility of our simulation model. Meaningful validation of predictions from modelled atherosclerotic plaque model typically requires examination of bona fide atherosclerotic lesions. To analyze a more accurate plaque rupture, fluid-structure interaction is applied to three-dimensional blood-vessel carotid bifurcation modelling. A patient-specific pressure variation is applied onto the plaque to influence its vulnerability.

Results

Modelling of the human atherosclerotic artery with varying degrees of lipid core elasticity, fibrous cap thickness and calcification gap, which is defined as the distance between the fibrous cap and calcification agglomerate, form the basis of our rupture analysis. Finite element analysis shows that the calcification gap should be conservatively smaller than its threshold to maintain plaque stability. The results add new mechanistic insights and methodologically sound data to investigate plaque rupture mechanics.

Conclusion

Structural analysis using a three-dimensional calcified model represents a more realistic simulation of late-stage atherosclerotic plaque. We also demonstrate that increases of calcium content that is coupled with a decrease in lipid core volume can stabilize plaque structurally.     

Synergistic activity of hydrophilic modification in antibiotic polymers

Quaternized poly(vinylpyridine) is known to kill up to 99% of drug-resistant gram-positive and -negative bacteria but shows minimal biocompatibility. We report enhanced bactericidal activity of vinylpyridine through copolymerization with hydroxyethyl methacrylate and poly(ethylene gycol) methyl ether methacrylate. Copolymers with increasing comonomer content were synthesized by radical polymerization and quaternized with hexylbromide. We assessed the effects of the changes in polymer composition on the bactericidal activity of the surface activity using a bioluminescent pathogenic strain of Escherichia coli (O157:H7). By recording the photoluminescence emitted by these bacteria in contact with the copolymers, it was shown that several of the copolymers possess better antibacterial efficiency than quaternized poly(vinylpyridine). Results indicate that several of the copolymers synthesized possess antibacterial activity approximately 20 times greater than the pure quaternized poly(vinylpyridine) homopolymer, while only containing 1 wt % hexylated pyridinium. This behavior is explained by the increased surface wettability of the copolymers containing lesser amounts of poly(vinylpyridine), as bactericidal behavior correlates to the hydrophilicity of the system as measured by contact angles. A hydrophilicity based design-paradigm can significantly improve both the efficacy and the biocompatibility of antibacterial materials.     

Regulation of adipose tissue inflammation and systemic metabolism in murine obesity by polymer implants loaded with lentiviral vectors encoding human interleukin-4

Dysfunctional adipose tissue plays a central role in the pathogenesis of the obesity-related metabolic disease, including type 2 diabetes. Targeting adipose tissue using biopolymer implants is a novel therapeutic approach for metabolic disease. We transplanted porous poly(lactide-co-glycolide) (PLG) implants coated with human interleukin-4 (hIL-4)-expressing lentivirus into epididymal white adipose tissue (eWAT) of mice fed a high-fat diet. Tissue and systemic inflammation and metabolism were studied with flow cytometry, immunohistochemistry, quantitative real-time polymerase chain reaction, adipose tissue histology, and in vivo glucose tolerance testing at 2 and 10 weeks of a high-fat diet. PLG implants carrying hIL-4-expressing lentivirus implanted into epididymal white adipose tissue of mice-regulated adipose tissue inflammation, including increased CD3⁺CD4⁺ T-cell frequency, increased eWAT adipocyte hypertrophy, and decreased FASN and ATGL expression, along with reduced fasting blood glucose levels. These effects were observed in early obesity but were not maintained in established obesity. Local delivery of bioimplants loaded with cytokine-expressing lentivirus vectors to adipose tissue influences tissue inflammation and systemic metabolism in early obesity. Further study will be required to show more durable metabolic effects. These data demonstrate that polymer biomaterials implanted into adipose tissue have the potential to modulate local tissue and systemic inflammation and metabolism.     

Development patterns in young conifer-hardwood forests of interior Alaska

Abstract. The age structure and growth patterns of 53 young conifer-hardwood stands on upland, south-facing sites of interior Alaska were analyzed to determine the length of time for stand establishment after disturbance, the composition of early-successional stands compared to existing stands, and the potential for late-successional stands dominated by conifers. Mixed stands of Picea glauca, Populus tremuloides and Betula papyrifera represented five plant community types and developed as single cohorts after stand-replacement fires. In the Populus tremuloides/Arctostaphylos uva-ursi and Populus tremuloides/Shepherdia canadensis community types, hardwoods established rapidly and Picea glauca established slowly. In contrast, stands in the Betula papyrifera-Populus tremuloides/Viburnum edule, Betula papyrifera-Populus tremuloides/Alnus crispa, and Picea glauca-Betula papyrifera/Hylocomium splendens community types generally developed as a result of rapid, concurrent establishment of conifers and hardwoods. These single-cohort, mixed species development patterns are not consistent with continual establishment of conifers and are likely the result of unique life-history traits and frequent stand-replacement fires.     

Residues Distal from the Active Site that Alter Enzyme Function in M.HhaI DNA Cytosine Methyltransferase

Abstract

Ten M.HhaI residues were replaced with alanine to probe the importance of distal protein elements to substrate/cofactor binding21,methyl transfer, and product release. The substitutions, ranging from 6–20 Å from the active site were evaluated by thermodynamic analysis, pre-steady and steady-state kinetics, to obtain K_d ^AdoMet, K_d ^DNA, k_cat/K_m ^DNA, k_cat, and k_{methyltransfer} values. For the wild-type M.HhaI, product release steps dominate catalytic turnover while the 4-fold faster internal microscopic constant k_{methyltransfer} presents an upper limit. The methyl transfer reaction has δH? and δS? values of 10.3 kcal/mol and—29.4 cal/(mol K), respectively, consistent with a compressed transition state similar to that observed in the gas phase. Although the ten mutants remained largely unperturbed in methyl transfer, long-range effects influencing substrate/cofactor binding and product release were observed. Positive enhancements were seen in Asp⁷³Ala, which showed a 25fold improvement in AdoMet affinity and in Val²⁸²Ala, which showed a 4-fold improvement in catalytic turnover. Based on an analysis of the positional probability within the C⁵- cytosine DNA methyltransferase family we propose that certain conserved distal residues may be important in mediating long-range effects.