Actin-filled nuclear invaginations indicate degree of cell de-differentiation

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.     

Multi-Scale Biomechanical Remodeling in Aging and Genetic Mutant Murine Mitral Valve Leaflets: Insights into Marfan Syndrome

Mitral valve degeneration is a key component of the pathophysiology of Marfan syndrome. The biomechanical consequences of aging and genetic mutation in mitral valves are poorly understood because of limited tools to study this in mouse models. Our aim was to determine the global biomechanical and local cell-matrix deformation relationships in the aging and Marfan related Fbn1 mutated murine mitral valve. To conduct this investigation, a novel stretching apparatus and gripping method was implemented to directly quantify both global tissue biomechanics and local cellular deformation and matrix fiber realignment in murine mitral valves. Excised mitral valve leaflets from wild-type and Fbn1 mutant mice from 2 weeks to 10 months in age were tested in circumferential orientation under continuous laser optical imaging. Mouse mitral valves stiffen with age, correlating with increases in collagen fraction and matrix fiber alignment. Fbn1 mutation resulted in significantly more compliant valves (modulus 1.34±0.12 vs. 2.51±0.31 MPa, respectively, P<.01) at 4 months, corresponding with an increase in proportion of GAGs and decrease in elastin fraction. Local cellular deformation and fiber alignment change linearly with global tissue stretch, and these slopes become more extreme with aging. In comparison, Fbn1 mutated valves have decoupled cellular deformation and fiber alignment with tissue stretch. Taken together, quantitative understanding of multi-scale murine planar tissue biomechanics is essential for establishing consequences of aging and genetic mutations. Decoupling of local cell-matrix deformation kinematics with global tissue stretch may be an important mechanism of normal and pathological biomechanical remodeling in valves.     

A functional and structural investigation of the human fronto-basal volitional saccade network

Almost all cortical areas are connected to the subcortical basal ganglia (BG) through parallel recurrent inhibitory and excitatory loops, exerting volitional control over automatic behavior. As this model is largely based on non-human primate research, we used high resolution functional MRI and diffusion tensor imaging (DTI) to investigate the functional and structural organization of the human (pre)frontal cortico-basal network controlling eye movements. Participants performed saccades in darkness, pro- and antisaccades and observed stimuli during fixation. We observed several bilateral functional subdivisions along the precentral sulcus around the human frontal eye fields (FEF): a medial and lateral zone activating for saccades in darkness, a more fronto-medial zone preferentially active for ipsilateral antisaccades, and a large anterior strip along the precentral sulcus activating for visual stimulus presentation during fixation. The supplementary eye fields (SEF) were identified along the medial wall containing all aforementioned functions. In the striatum, the BG area receiving almost all cortical input, all saccade related activation was observed in the putamen, previously considered a skeletomotor striatal subdivision. Activation elicited by the cue instructing pro or antisaccade trials was clearest in the medial FEF and right putamen. DTI fiber tracking revealed that the subdivisions of the human FEF complex are mainly connected to the putamen, in agreement with the fMRI findings. The present findings demonstrate that the human FEF has functional subdivisions somewhat comparable to non-human primates. However, the connections to and activation in the human striatum preferentially involve the putamen, not the caudate nucleus as is reported for monkeys. This could imply that fronto-striatal projections for the oculomotor system are fundamentally different between humans and monkeys. Alternatively, there could be a bias in published reports of monkey studies favoring the caudate nucleus over the putamen in the search for oculomotor functions.     

Cytoskeletal heart-enriched actin-associated protein (CHAP) is expressed in striated and smooth muscle cells in chick and mouse during embryonic and adult stages

We recently identified a new Z-disc protein, CHAP (Cytoskeletal Heart-enriched Actin-associated Protein), which is expressed in striated muscle and plays an important role during embryonic muscle development in mouse and zebrafish. Here, we confirm and further extend these findings by (i) the identification and characterization of the CHAP orthologue in chick and (ii) providing a detailed analysis of CHAP expression in mouse during embryonic and adult stages. Chick CHAP contains a PDZ domain and a nuclear localization signal, resembling the human and mouse CHAPa. CHAP is expressed in the developing heart and somites, as well as muscle precursors of the limb buds in mouse and chick embryos. CHAP expression in heart and skeletal muscle is maintained in adult mice, both in slow and fast muscle fibers. Moreover, besides expression in striated muscle, we demonstrate that CHAP is expressed in smooth muscle cells of aorta, carotid and coronary arteries in adult mice, but not during embryonic development.     

Antisense oligonucleotide reduction of apoB-ameliorated atherosclerosis in LDL receptor-deficient mice

Chronic elevations of plasma apolipoprotein B (apoB) are strongly associated with cardiovascular disease. We have previously demonstrated that inhibition of hepatic apoB mRNA using antisense oligonucleotides (ASO) results in reductions of apoB, VLDL, and LDL in several preclinical animal models and humans. In this study, we evaluated the anti-atherogenic effects of a murine-specific apoB ASO (ISIS 147764) in hypercholesterolemic LDLr deficient (LDLr(-/-)) mice. ISIS 147764 was administered weekly at 25-100 mg/kg for 10-12 weeks and produced dose-dependent reductions of hepatic apoB mRNA and plasma LDL by 60-90%. No effects on these parameters were seen in mice receiving control ASOs. ApoB ASO treatment also produced dose-dependent reductions of aortic en face and sinus atherosclerosis from 50-90%, with high-dose treatment displaying less disease than the saline-treated, chow-fed LDLr(-/-) mice. No changes in intestinal cholesterol absorption were seen with apoB ASO treatment, suggesting that the cholesterol-lowering pharmacology of 147764 was primarily due to inhibition of hepatic apoB synthesis and secretion. In summary, ASO-mediated suppression of apoB mRNA expression profoundly reduced plasma lipids and atherogenesis in LDLr(-/-) mice, leading to the hypothesis that apoB inhibition in humans with impaired LDLr activity may produce similar effects.     

Genome-wide association study identifies a single major locus contributing to survival into old age; the APOE locus revisited

By studying the loci that contribute to human longevity, we aim to identify mechanisms that contribute to healthy aging. To identify such loci, we performed a genome-wide association study (GWAS) comparing 403 unrelated nonagenarians from long-living families included in the Leiden Longevity Study (LLS) and 1670 younger population controls. The strongest candidate SNPs from this GWAS have been analyzed in a meta-analysis of nonagenarian cases from the Rotterdam Study, Leiden 85-plus study, and Danish 1905 cohort. Only one of the 62 prioritized SNPs from the GWAS analysis (P<1×10(-4) ) showed genome-wide significance with survival into old age in the meta-analysis of 4149 nonagenarian cases and 7582 younger controls [OR=0.71 (95% CI 0.65-0.77), P=3.39 × 10(-17) ]. This SNP, rs2075650, is located in TOMM40 at chromosome 19q13.32 close to the apolipoprotein E (APOE) gene. Although there was only moderate linkage disequilibrium between rs2075650 and the ApoE ε4 defining SNP rs429358, we could not find an APOE-independent effect of rs2075650 on longevity, either in cross-sectional or in longitudinal analyses. As expected, rs429358 associated with metabolic phenotypes in the offspring of the nonagenarian cases from the LLS and their partners. In addition, we observed a novel association between this locus and serum levels of IGF-1 in women (P=0.005). In conclusion, the major locus determining familial longevity up to high age as detected by GWAS was marked by rs2075650, which tags the deleterious effects of the ApoE ε4 allele. No other major longevity locus was found.     

Maternal Smoking during Pregnancy and Fetal Organ Growth: A Magnetic Resonance Imaging Study

Objective

To study whether maternal cigarette smoking during pregnancy is associated with alterations in the growth of fetal lungs, kidneys, liver, brain, and placenta.

Design

A case-control study, with operators performing the image analysis blinded.

Setting

Study performed on a research-dedicated magnetic resonance imaging (MRI) scanner (1.5 T) with participants recruited from a large teaching hospital in the United Kingdom.

Participants

A total of 26 pregnant women (13 current smokers, 13 non smokers) were recruited; 18 women (10 current smokers, 8 nonsmokers) returned for the second scan later in their pregnancy.

Methods

Each fetus was scanned with MRI at 22–27 weeks and 33–38 weeks gestational age (GA).

Main outcome measures

Images obtained with MRI were used to measure volumes of the fetal brain, kidneys, lungs, liver and overall fetal size, as well as placental volumes.

Results

Exposed fetuses showed lower brain volumes, kidney volumes, and total fetal volumes, with this effect being greater at visit 2 than at visit 1 for brain and kidney volumes, and greater at visit 1 than at visit 2 for total fetal volume. Exposed fetuses also demonstrated lower lung volume and placental volume, and this effect was similar at both visits. No difference was found between the exposed and nonexposed fetuses with regards to liver volume.

Conclusion

Magnetic resonance imaging has been used to show that maternal smoking is associated with reduced growth of fetal brain, lung and kidney; this effect persists even when the volumes are corrected for maternal education, gestational age, and fetal sex. As expected, the fetuses exposed to maternal smoking are smaller in size. Similarly, placental volumes are smaller in smoking versus nonsmoking pregnant women.